Note: Descriptions are shown in the official language in which they were submitted.
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Peptide reagent for the detection of human cytomegalovirus (CMV).
. The present invention relates to a peptide reagent comprising peptides
immunochemically reactive with antibodies to the human cytomegalovirus {CMV),
(monoclonal) antibodies directed against said peptides, and cell lines capable
of producing
monoclonal antibodies. The invention is further concerned with methods for
(direct)
detection of CMV or (indirect) detection of antibodies directed against CMV.
Cytomegalovirus (CMV) is a world-wide spread member of the human herpesvirus
l0 family, infecting between 50-100% of all individuals depending on age and
socio-economic
Stahl S.
CMV is naturally transmitted via saliva, urine or breast-milk but can also be
recovered from other body secretions. In addition, CMV can be transmitted
transplacentally to the foetus, by geno-urinary contact during birth or sexual
intercourse,
by blood transfusion (esp. white cells) and bone marrow cq. organ
transplantation.
After primary infection CMV persists in the body for the lifetime of its host
in a
state of dynamic latency, well controlled by the host immune system and may be
recovered
periodically from different sites and body secretions.
Although generally benign, CMV infections can be devastating and fatal in
2 0 individuals with immune defects, such as transplant recipients, AIDS-
patients, patients
with genetically determined immunodeficiencies and newborns with an immature
immune
system.
Therapeutic intervention is possible using drugs affecting viral DNA-
replication but
this is associated with significant toxicity. Modulation of host immunity is
another
2 ~ effective way of controlling CMV infection, which can be achieved by
vaccination, passive
immunotherapy using immunoglobulins or T-cells or by manipulation of
immunosuppressive therapy.
Identification and monitoring of active (symptomatic) infection due to CMV
cannot
be done based upon clinical parameters alone, - as these are diverse and
variable-, but
30 heavily depends on rapid and accurate laboratory diagnosis.
CMV-specific diagnosis can be achieved by a variety of techniques directly
detecting viral components or indirectly measuring changes in the hosts immune
status.
Reliable diagnostic approaches require sensitive and reproducible technology
based upon
well defined and highly CMV-specific reagents and a detailed understanding of
the
35 molecular processes underlying CMV-infection in the human host.
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CMV is the largest and most complex of the human herpesvirinae, with a genome
of 230-270 kilobases.
Characteristic for a herpesvirus, the structural components of the CMV virion
include a protein-DNA core, an icosahedral capsid consisting of 162 capsomer
subunits, an
amorphous layer of proteins called tegument and a surrounding proteo-Iipid
envelope
essential for virus infectivity.
Although in vivo CMV can be found in a variety of host cells and tissues, with
different levels of viral gene expression, efficient gene-expression and viral
DNA-
replication in vitro is only possible in primary fibroblast cells of human
origin. Therefore
this system has been most widely used to study viral gene expression,
replication and
characterization of related protein products.
Following binding, de-envelopment and penetration into a susceptible host cell
(e.g.
human fibroblast), tegument proteins from the incoming virion activate the
expression of
viral genes of the immediate early (IEA) class, encoding a limited number of
protein
species with broad-acting transcriptional activating functions, capable of
transactivating the
transcription of a wide variety of viral and host genes. Viral genes activated
by IEA
products predominantly consist of enzymes involved in nucleotide metabolism
and DNA-
synthesis and are classified as the group of early antigens (EA). In the last
stage of
infection and essentially depending on the synthesis of new viral DNA-
templates by the
virally encoded DNA-polymerase (an EA component), a third (late antigen (LA))
group of
viral proteins is expressed, consisting of the structural components required
for assembly
and release of viral progeny.
Both in vivo and in vitro, this sequence of gene-expression can be interrupted
at
various stages leading to non-lyric (abortive or defective) infection cq.
persistence, which
2 5 is characterized by restricted gene-expression patterns. These patterns
are both dependent
on the type of cell infected and on its activation and differentiation status.
Viral gene
products thus expressed still may alter host cell characteristics resulting in
aberrant
behavior and function.
In addition to the expression of its own genes in a 3-step cascade regulated
fashion,
3 0 CMV stimulates host-cell gene expression following infection, thus
complicating the
specific analysis and purification of viral products.
In blood, CMV can be found in sporadic monocyte/macrophages (replicating),
lymphocytes (abortive/restricted), polymorph nuclear cells (only PP65 protein)
and
35 circulating endothelial cells (replicating). Furthermore CMV can be
detected in smooth
muscle cells lining the arteries and smaller blood vessels (restricted/lytic)
and fibroblast
and macrophage-like cells in a variety of tissues.
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In the absence of effective immune responses CMV can spread to virtually any
tissue and cell type in the body.
The presence of infectious virus in the blood as determined by culture
techniques is
considered the best marker associated with active symptomatic infection,
whereas the
presence of viral DNA is a marker for virus carrier status.
CMV is associated with a wide variety of disease syndromes both in the
immunocompetent and in the immunocompromised host, although the latter are
much more
frequent and associated with significantly greater morbidity and mortality.
Primary infection in the immunocompetent host usually go unnoticed. However
CMV is considered to be causing 10% of the mononucleosis syndrome in
adolescents and
young adults and is frequently associated with acute nonA-G hepatitis. Primary
infection
in pregnant women is associated with the transplacental transfer of CMV to the
foetus.
CMV is the leading cause of birth defects in the world, infecting between 0.5
and
3% of all newborns. In about 10% of infected newborns CMV-related defects can
be
detected, which are most serious in case of a primary infection in the mother.
CMV is named the "Troll of transplantation" as it is the most frequent
infectious
cause of complications in solid organ and bone-marrow transplant recipients.
CMV also is a leading cause of disease in HIV positives and AIDS-patients,
most
2 0 frequently associated with pneumonia, retinitis and gastro-intestinal
complications.
Finally, CMV frequently causes disease in patients with genetically determined
immuno-deficiencies, cancer patients and patients with autoimmune diseases.
The quality of the host immune status determines the balance between CMV
replication and spread and viral persistence in a dormant latency state.
Host immune responses are induced and maintained upon encounter of newly
synthesized or stable persisting viral gene products expressed during the
different stages of
infection at various sites/tissues in the body.
Depending on virus-host interactions at the molecular level -which may be
different
for each individual- immune responses are build-up gradually following primary
infection
until a state of balance is achieved, which is called latency.
Determination of the quality and quantity of host immune responses to CMV are
of
diagnostic and prognostic importance and have been widely used to determine
immune
status to CMV, to establish the donor/recipient CMV carrier status in the
transplant setting
3 5 and to identifylmonitor acute infections in a variety of CMV-associated
disease syndromes.
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Diagnostic assays may directly measure the presence and quantity of virus in
body
fluids, by means of virus culture or DNA detection/quantitation techniques..
However,
only CMV detection in the blood is considered as a reliable parameter for
diagnosis of
active infection in man, as the virus may be secreted intermittently in urine
and saliva in
most healthy CMV carriers.
DNA-detection in blood is not considered a reliable predictor for active CMV-
infection as latently infected CMV-DNA positive leukocytes can be detected in
most if not
all healthy carriers, depending on the sensitivity of the assay used.
Measurement of CMV-RNA expression in blood leukocytes may be useful for
detecting active virus replication in the blood depending on the genomic
target sequence
analyzed.
Alternatively the antigenaemia assay, i.e. the quantitation of blood
leukocytes (esp.
polymorph nuclear cells) for the presence of intracellular PP65{UL83), has
proven to be a
reliable diagnostic parameter correlating well with active and symptomatic CMV
infection
in a variety of CMV disease syndromes.
In contrast to methods for the direct detection of virus or viral products,
the
analysis of humoral immune responses to CMV can be used as an indirect
reflection of
CMV activity in the human host. CMV serology is probably the most widely used
approach for the diagnosis and monitoring of active CMV infections and for
determination
2 0 of CMV carrier status. A wide variety of techniques are available which
are both rapid and
cheap for detecting IgM, IgG or IgA antibodies to CMV.
Whereas the detection of CMV-IgM and to a lesser extend CMV-IgA is directly
indicative for (recent) active infection, -especially during a primary
infection-, the
measurement of CMV-IgG may be applied both for determination of CMV carrier
status
~ 5 and for diagnosing/monitoring CMV-disease.
During primary infection IgG antibodies are formed rapidly and remain present
for
life. Besides the presence of CMV-IgM a significant rise in CMV-IgG is
reflecting active
infection in the host. The latter may be complicated in case of full blood
transfusion or
immunoglobulin therapy.
3 0 Besides being a measure of active infection the generation of an antibody
response
to CMV is a reflection of immunocompetence and can be used to guide anti-viral
therapy
in the immunocompromised. Detection of a brisk antibody response during
antiviral
therapy may be used as a sign to end the therapy and to allow the immune
system to take
over the control of CMV in the host.
35 Thus, serology is a reliable and versatile tool in the diagnosis and
monitoring of
CMV infections in the human host.
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In the design of serological assays a crucial ingredient is the CMV-specific
antigen
preparation which serves to bind immunoglobulins in the sample to be analyzed.
Thus it is
important to define the molecular fine specificity of anti-CMV antibody
responses in order
' to allow standardization. Such studies have been initiated in recent years
but have proven
5 difficult due to the diversity of anti-CMV antibody responses and the
complexity of the
CMV system.
Although the 235KB CMV genome of prototype strain AD169 has been sequenced
completely and over 200 potential protein encoding open reading frames have
been
identified, only some 40 of these proteins have been biochemically and
immunologically
1 o studied to date. A limited number of CMV-polypeptides have been defined as
target for
human antibody responses, such as pp150 (UL32), pp72(UL1221123}, pp65(UL83),
pp52(UL44), pp38(UL80), pp28(UL99), gB(UL55} and MDBP(UL57). Additional
immunoreactive polypeptides have been defined by their molecular weight in CMV
infected cell extracts analyzed using the immunoblot technique, but their
coding frames on
the CMV genome have not been defined yet.
As stated above many different techniques are currently used for CMV serology.
These methods either use cell culture derived (semi-purified) antigen
extracts,
partially purified extracellular virions and dense bodies or more defined
(recombinant)
2 o proteins and fragments thereof. Due to this variation of methodology and
lack of uniform
and well defined reagents, CMV-serology currently is not well standardized.
Reliable
diagnosis and further standardization of CMV serology therefore calls for the
use of
molecular defined and highly purified CMV antigens.
Such antigens may be produced and purified from CMV cell culture, but this is
2 5 expensive and complicated by the presence of a multitude of host cell
proteins.
Recombinant CMV proteins expressed in alternative host systems require even
higher
grade of purification as patient sera may have antibodies to such host cell
components
leading to potential false positive results.
An example of the use of recombinant proteins can be found in Patent
Application
30 WO 95/00073, wherein a mixture of recombinant antigens (fusion proteins)
are used to
detect CMV specific IgM.
Synthetic peptides represent a highly defined alternative for such protein
antigens
and can be produced and purified in a highly reproducible manner.
It is an object of this invention to define an optimal combination of
synthetic
peptide reagents that are specifically suited for application in CMV
serological techniques
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as they are highly reactive with human serum immunoglobulins
of both the IgG and IgM class.
These reagents can be synthesized highly
reproducible and are easily purified, thus well suited for
further improvement and standardization of CMV-serology.
Antibodies to these peptide reagents may be useful
in the development and quality control of serologic assays
and for the direct detection of CMV.
Defining synthetic peptide fragments, representing
immunodominant domains of CMV-proteins, capable of replacing
the intact proteins in diagnostic tests, is a subject of the
present invention.
Synthetic peptides have the advantage of being
chemically well defined, thus allowing easy and reproducible
production at high yields, well suited for application in
diagnostic assays which can be manufactured and used with
greater reproducibility.
The present invention provides a peptide reagent
for the detection of antibodies to the Cytomegalovirus,
wherein said reagent comprises a peptide derived from the
Cytomegalovirus structural phosphoprotein pp150 (UL32) and a
peptide derived from one of the Cytomegalovirus proteins;
pp52 (UL44), pp28 (UL99), or gB (UL55).
Thus in one aspect, the present invention provides
a peptide reagent comprising: (a) at least one first
synthetic peptide reactive with antibodies to
Cytomegalovirus, said first synthetic peptide comprising:
(i) an amino acid sequence selected from the group
consisting of SEQ ID NOs:l, 2, 3, 4, 5, and 6; or (ii) an
immunogenic fragment of the amino acid sequence in (a)(i);
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and (b) at least one second synthetic peptide reactive with
antibodies to Cytomegalovirus, said second synthetic peptide
comprising: (i) an amino acid sequence selected from the
group consisting of SEQ ID NOs:7, 8, 9, and 10; or (ii) an
immunogenic fragment of the amino acid sequence in (b)(i).
In another aspect, the present invention provides
a method of detecting an antibody against Cytomegalovirus in
a sample, the method comprising the steps of: (a) contacting
said sample with the peptide reagent according to the
invention; and (b) detecting formation of immune complexes
formed in (a).
In another aspect, the present invention provides
use of the peptide reagent according to the invention for
the detection of antibodies against Cytomegalovirus.
In another aspect, the present invention provides
a test kit for carrying out the method according to the
invention, comprising the peptide reagent according to the
invention together with instructions for use for detecting
an antibody against Cytomegalovirus.
Furthermore, the present invention provides a
peptide reagent comprising a peptide derived from CMV pp150-
protein comprising at least part of the amino acid sequence
as shown in SEQ ID No:l-6.
A preferred embodiment of the present invention is
a peptide reagent comprising a peptide derived from CMV
pp150-protein comprising at least part of the amino acid
sequence as shown in SEQ ID No: 1.
A further object of the present invention is a
peptide reagent comprising a peptide derived from CMV
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gB-protein comprising at least part of the amino acid
sequence as shown in SEQ ID No:7.
Another object of the present invention is a
peptide reagent comprising a peptide derived from CMV pp52-
protein comprising at least part of the amino acid sequence
as shown in SEQ ID No:8.
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Another object of the present invention is a peptide reagent comprising a
peptide
derived from CMV pp28-protein comprising at least part of the amino acid
sequence as
shown in SEQ.ID.No.: 9-10.
A preferred embodiment of the present invention is a peptide reagent
comprising a
peptide having the amino acid sequence as shown in SEQ 1D No.: 10.
A most preferred embodiment of the present invention is a peptide reagem
comprising a peptide having the amino acid sequence as shown in SEQ ID No.: 1,
a
peptide having the amino acid sequence as shown in SEQ ID No.: 7 and a peptide
having
the amino acid sequence shown in SEQ.ID. No.: 10.
Another most preferred embodiment of the present invention is a peptide
reagent
comprising a peptide having a amino acid sequence as shown in SEQ ID No.: l
and a
peptide having the amino acid sequence shown in SEQ.ID.No.: 8.
In contrast to the natural CMV, the peptides according to the invention have
the
great advantage that these are of a safe non-infectious origin.
2 0 The peptides and fragments thereof which are preferably used in a peptide
reagent
according to the invention are found to be particularly suitable for use in a
diagnostic
method for the determination of the presence of CMV or CMV-antibodies in a
sample.
Moreover, said peptides and fragments thereof may be used in suitable
pharmaceutical
dosage forms in the treatment of a CMV-related disease. The preparation of
vaccines thus
2 5 obtained which contain a peptide or fragment thereof as active
ingredients, is known to one
skilled in the art.
The peptides which are preferably used in a peptide reagent according to the
present invention have improved reactivity and specificity (performance)
compared with
30 currently available CMV reagents.
Therefore the utilization of these immunological reagents in serological tests
allows
the development of assays that will permit a better differential diagnosis in
patients with
active CMV-infections.
35 Furthermore, object of the present invention is the finding that the
presence of
antibodies to the selected CMV peptides is correlated with active CMV-
infection.
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The term "peptide reagent" as used herein refers to one or more peptides and a
suitable support or a labeling substance.
Supports which can be used are, for example, the inner wall of a microtest
well or
a cuvette, a tube or capillary, a membrane, filter, test strip or the surface
of a particle such
as, for example, a latex particle, an aldehyde particle (such as a ceramic
magnetizable
particle with active aldehyde surface groups), an erythrocyte, a dye sol, a
metal sol or
metal compound as sot particle, a carrier protein such as BSA or KLH.
Labeling substances which can be used are, inter aIia, a radioactive isotope,
a
fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol
particle.
l0 In a method for the detection of antibodies directed against CMV in a
sample, an
peptide reagent according to the invention is brought into contact with the
sample. After
which, the presence of immune complexes formed between the peptide and
antibodies in
the sample is detected and by this detection the presence of CMV antibodies in
the sample
is known and can be determined quantitatively.
Depending on the nature and further characteristics of the peptide reagent the
immunochemical reaction that takes place is a so called sandwich reaction, an
agglutination
reaction, a competition reaction or an inhibition reaction.
2 0 The term "peptide" as used herein refers to a molecular chain of amino
acids with a
biological activity, and does not refer to a specific length of the product.
Thus inter alia,
proteins, fusion-proteins or -peptides oligopeptides and polypeptides are
included.
If required peptides which are preferably used in a peptide reagent according
to the
invention can be modified in vivo or in vitro, for example by glycosylation,
amidation,
2 5 carboxylation or phosphorylation. Functional variants like, for example,
acid addition
salts, amides, esters, and specifically C-terminal esters, and N-acyl
derivatives of said
peptides are therefore also considered part of the present invention. It will
be understood
that for the particular proteins or polypeptides embraced herein, natural
variations can also
exist. These variations may be demonstrated by {an) amino acid differences) in
the overall
3 0 sequence or by deletions, substitutions, insertions, inversions or
additions of (an) amino
acids) in said sequence. Amino acid substitutions from which can be expected
that they do
not essentially alter biological and immunological activities, have been
described. Amino
acid replacements between related amino acids or replacements which have
occurred
frequently in evolution are, inter alia Ser/Ala, SerIGly, Asp/Gly, AsplAsn,
IIe/Val (see
35 Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res.
Found.,
Washington D.C., 1978, vol. 5, suppl. 3). Based on this information Lipman and
Pearson
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developed a method for rapid and sensitive protein comparison (Science 227,
1435-1441,
1985) and determining the functional similarity between homologous proteins.
The term "at least a part of" as used herein means an amino acid sequence
comprising a subsequence of a peptide which is preferably used in a peptide
reagent
according to the invention. Said part or fragment is a peptide having one or
more
immunogenic determinants of the CMV pp28 (UL99), pp150 (UL32),
pp72(UL122/123),
pp65(UL83), pp52(UL44), pp38(UL80), gB(UL55) and MDBP(UL57) proteins.
Fragments can inter alia be produced by enzymatic cleavage of precursor
molecules, using
restriction endonucleases for the DNA and proteases for the polypeptides.
Other methods
include chemical synthesis of the fragments or the expression of peptide
fragments by
DNA fragments.
Suitable antigenic or immunogenic fragments of said peptides containing (an)
epitope(s) can be found by means of the method described in European Patent EP
0 220
I5 245, Geysen, H.M. et al. (Proc. Natl. Acad. Sci. 81, 3998-4002, 1984),
Geysen, H.M. et
al. (J. Immunol. Meth. 102, 259-274, 1987) based on the so-called pepscan
method,
wherein a series of partially overlapping peptides corresponding with partial
sequences of
the complete polypeptide under consideration, are synthesized and their
reactivity with
antibodies is investigated.
2 0 In addition, a number of regions of the peptides can be designated
epitopes on the
basis of theoretical considerations, although the predictive value of these
theoretical
considerations is limited. The determination of these regions is based on a
combination of
the hydrophilicity criteria according to Hopp and Woods (Proc. Natl. Acad.
Sci. 78, 3824
3828, 1981) and the secondary structure aspects according to Chou and Fasman
(Advances
25 in Enzymology 47, 45-148, 1987).
The preparation of the peptides or fragments thereof that are preferably used
in a
peptide reagent according to the invention is effected by means of one of the
known
organic chemical methods for peptide synthesis or with the aid of recombinant
DNA
3 0 techniques.
The organic chemical methods for peptide synthesis are considered to include
the
coupling of the required amino acids by means of a condensation reaction,
either in
homogeneous phase or with the aid of a so-called solid phase.
35 The condensation reaction can be carried out as follows:
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a) condensation of a compound (amino acid, peptide) with a free carboxyl group
and protected other reactive groups with a compound (amino acid, peptide) with
a free
amino group and protected other reactive groups, in the presence of a
condensation agent;
b) condensation of a compound (amino acid, peptide) with an activated carboxyl
5 group and free or protected other reaction groups with a compound (amino
acid, peptide)
with a free amino group and free or protected other reactive groups.
Activation of the carboxyl group ca.n take place, inter alia, by converting
the
carboxyl group to an acid halide, azide, anhydride, imidazolide or an
activated ester, such
as the N-hydroxy-succinimide, N-hydroxy-benzotriazole or p-nitrophenyl ester.
The most common methods for the above condensation reactions are: the
carbodiimide method, the azide method, the mixed anhydride method and the
method using
activated esters, such as described in The Peptides, Analysis, Synthesis,
Biology Vol. 1-3
(Ed. Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc.).
Preparation of suitable fragments of above-mentioned peptides using the "solid
phase method" is for instance described in J. Amer. Chem. Soc. 85, 2149 (1963)
and Int.
J. Peptide Protein Res. 35, 161-214 (1990). The coupling of the amino acids of
the peptide
to be prepared usually starts from the carboxyl end side. For this method a
solid phase is
2 0 needed on which there are reactive groups or on which such groups can be
introduced.
This can be, for example, a copolymer of benzene and divinylbenzene with
reactive
chloromethyl groups, or a polymeric solid phase rendered reactive with
hydroxymethyl or
amine-function.
A particularly suitable solid phase is, for example, the p-alkoxybenzyl
alcohol resin
2 5 (4-hydroxy-methyl-phenoxy-methyl-copolystrene-1 % divinyl-benzene resin),
described by
Wang (1974; J. Am. Chem. Soc. 95, 1328). After synthesis the peptides can be
split from
this solid phase under mild conditions.
After synthesis of the desired amino acid sequence, detaching of the peptide
from
the resin follows, for example, with trifluoromethane-sulphonic acid or with
3 0 methanesulphonic acid dissolved in trifluoroacetic acid. The peptide can
also be removed
from the carrier by transesterification with a lower alcohol, preferably
methanol or
ethanol, in which case a tower alkyl ester of the peptide is formed directly.
Likewise,
splitting with the aid of ammonia gives the amide of a peptide according to
the invention.
35 The reactive groups which may not participate in the condensation reaction
are, as
stated, effectively protected by groups which can be removed again very easily
by
hydrolysis with the aid of acid, base or reduction. Thus, a carboxyl group can
be
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effectively protected by, for example, esterification with methanol, ethanol,
tertiary
butanol, benzyl alcohol or p-nitrobenzyl alcohol and amines linked to solid
support.
Groups which can effectively protect an amino group are the ethoxycarbonyl,
benzyioxycarbonyl, t-butoxy-carbonyl (t-boc) or p-methoxy-benzyloxycarbonyl
group, or
an acid group derived from a sulphonic acid, such as the benzene-sulphonyl or
p-toluene-
sulphonyl group, but other groups can also be used, such as substituted or
unsubstituted
aryl or aralkyl groups, for example benzyl and triphenylmethyl, or groups such
as ortho-
nitrophenyl-sulphenyl and 2-benzoyl-1-methyl-vinyl. A particularly suitable A-
amino-
l0 protective group is, for example, the base-sensitive 9-fluorenyl-
methoxycarbonyl (Fmoc)
group [Carpino & Han (1970) J. Amer. Chem. Soc. 92, 5748].
A more extensive account of possible protecting groups can be found in The
Peptides, Analysis, Synthesis, Biology, Vol. 1 - 9 (Eds. Gross, Udenfriend and
Meienhofer) 1979 - 1987 (Academic Press, Inc.).
It is necessary also to protect the E-amino group of lysine and advisable for
the
guanidine group of arginine. Customary protective groups in this connection
are a Boc-
group for lysine and a Pmc- or Pms- or Mbs-group or Mtr-group for arginine.
The protective groups can be split off by various conventional methods,
depending
on the nature of the particular group, for example with the aid of
trifluoroacetic acid or by
mild reduction, for example with hydrogen and a catalyst, such as palladium,
or with HBr
in glacial acetic acid.
The immunoreactive peptides that are used in a peptide reagent according to
the
present invention can also be combined in a single molecule. The covalent
linkage of two
or more peptides in a hybrid- or combi-peptide can for instance be carried out
through
solid phase peptide synthesis, using the methods described above, of a peptide
sequence
wherein the amino acid sequences of the individual peptides are aligned. It is
understood
that a linker sequence may be inserted between the individual peptides
sequences. Such a
linker sequence may for instance be a stretch of 2-5 residues of glycine.
A hybrid- or combi-peptide can also be prepared through solid phase synthesis
using the fragment condensation approach. The latter method, in which the
fragments (the
sequences of which may correspond with the sequences of the individual
peptides of the
invention) are separately prepared and purified, is preferred in the synthesis
of the longer
hybrid- or combi-peptide sequences. The methodology for the preparation of
longer
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peptides is known in the art, and for instance described in The Peptides,
Analysis,
Biology, Vol. 1-9 (vide supra).
Alternatively, hybrid- or combi-peptides can be prepared through conjugation
of
appropriately modified peptides of the present invention.
In a preferred method for the conjugation of two different peptide sequences
which
are devoid of the amino acid cysteine, the peptides are derivatized to contain
an additional
residue of cysteine at either the carboxyl- or the amino-terminal end. One of
the peptides is
subsequently activated at the single cysteine thiol function with 2,2'-
dithiodipyridine. The
resulting pyridyl-dithio-peptide derivative is then reacted with the second
peptide
containing the cysteine thiol group to yield a hybrid peptide in which the
individual
peptides are linked through a disulfide bond.
Numerous other methods for the preparation of hybrid peptides can be
envisaged.
Use can be made of the chemical methodology that has been developed in the
field of
protein-protein conjugation. An overview of such methods is given by Means and
Feeney
(Bioconj. Chem. 1, 2-12, 1990). For instance, the use of well known homo- or
heterobifunctional cross-linking agents allow the coupling of individual
peptides through a
disulfide bond, or a thioether or amide bond, or the like.
As already indicated above, the peptides that are preferably used in a peptide
2 0 reagent according to the invention can likewise be prepared with the aid
of recombinant
DNA techniques. This possibility is of importance particularly when the
peptide is
incorporated in a repeating sequence ("in tandem ") or when the peptide can be
prepared as
a constituent of a (much larger) protein or polypeptide or as a fusion protein
with, for
example, (part of) (3-galactosidase. This type of peptides therefore likewise
falls within the
2 5 scope of the invention. For this purpose, as a constituent of a
recombinant DNA, a nucleic
acid sequence is used which codes for a peptide according to the invention and
which,
furthermore, is substantially free from nucleic acid segments, which in the
naturally
occurring CMV genome flank the nucleic acid sequence indicated above.
3 0 This latter method involves the preparation of the desired peptide by
means of
bringing to expression a recombinant polynucleotide with a nucleic acid
sequence which is
coding for one or more of the peptides in question in a suitable micro-
organism as host.
A nucleic acid sequence encoding a peptide as used in a peptide reagent
according
3 5 to the present invention can be ligated to various replication effecting
DNA sequences with
which it is not associated or linked in nature resulting in a so called
recombinant vector
molecule which can be used for the transformation of a suitable host. Useful
recombinant
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13
vector molecules, are preferably derived from, for example plasmids,
bacteriophages,
cosmids or viruses.
Specific vectors or cloning vehicles which can be used to clone nucleic acid
sequences are known in the art and include inter alia plasmid vectors such as
pBR322, the
various pUC, pGEM and Bluescript plasmids, bacteriophages, e.g. kgt-Wes,
Charon 28
and the M13 derived phages or viral vectors such as SV40, adenovirus or
polyoma virus
{see also Rodriquez, R.L. and D.T. Denhardt, ed., Vectors: A survey of
molecular
cloning vectors and their uses, Butterworths, 1988; Lenstra, J.A. et al.,
Arch. Virol. 110,
1-24, 1990). The methods to be used for the construction of a recombinant
vector molecule
are known to those of ordinarily skill in the art and are inter alia set forth
in Maniatis, T.
et al. (Molecular Cloning A Laboratory Manual, second edition; Cold Spring
Harbor
Laboratory, 1989).
For example, the insertion of the nucleic acid sequence encoding a peptide
according to the invention into a cloning vector can easily be achieved when
both the genes
and the desired cloning vehicle have been cut with the same restriction
enzymes) as
complementary DNA termini are thereby produced.
The recombinant vector molecules may additionally contain one or more marker
activities that may be used to select for desired transformants, such as
ampicillin and
tetracycline resistance in pBR322, as for example ampicillin resistance and a-
peptide of 13
galactosidase in pUC8.
It should, of course, be understood that the nucleotide sequences inserted at
the
selected site of the cloning vector may include only a fragment of the
complete nucleic acid
sequence encoding for peptides used in a peptide reagent according to the
invention as long
2 5 as the transformed host will produce a polypeptide having at least one or
more antigenic or
immunogenic determinants.
Antibodies, directed to said peptides are also part of the present invention.
The peptides or fragments thereof prepared and described above can be used to
produce antibodies, both polyclonal and monoclonal.
The monoclonal antibodies according to the present invention, therefore,
provide a
new means for the diagnosis of CMV infection.
Preferred antibodies according to the invention are monoclonal antibodies
which
bind to an epitope of a peptide having the amino acid sequence as shown in SEQ
ID No.:
1-10.
More preferred antibodies according to the invention are monoclonal antibodies
which bind to an epitope of the CMV pp150-protein, which epitope is recognized
by
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monoclonal antibody CMV.OT3C produced by the hybridoma cell lines deposited
with the
European Collection of Animal Cell Cultures {ECACC), CAMR {Centre for Applied
Microbiology & Research), Salisbury {UK), under deposit No. 96071123.
Immortalized cell lines capable of excreting human or animal (e.g. mouse, rat
or
chimpanzee) monoclonal antibodies according to the invention are also part of
the present
invention.
The preparation of cell lines producing monoclonal antibodies may occur by,
for
example, by the Kohler and Milstein technique (Kohler and Milstein devised the
techniques
that resulted in the formation monoclonal antibody-producing hybridomas (G.
Kohler and
C. Milstein, 1975, Nature 256:495-497; 1976, Eur. J. Immunol. 6:511-519)),
transformation with Epstein-Barr Virus, or a direct transformation technique
of B
lymphocytes with oncogenic DNA, or a direct fusion of human B-lymphocytes with
a
fusion partner being either a human or a mouse-human hybrid myeloma cell line,
or a
direct fusion of an EBV-transformed B cell line with said myeloma cell lines.
Preferred cell lines according to the invention are the cell lines deposited
at the
European Collection of Animal Cell Cultures, CAMR (Centre for Applied
Microbiology &
Research), Salisbury (UK) under deposit No. 96071123.
2 0 These hybridoma cell lines were produced by the fusion of a myeloma cell
with a
lymphocyte derived from a mouse previously inoculated with CMV-related
synthetic
peptide molecules (if necessary coupled to keyhole limpet hemacyanin (KLH) in
order to
enhance their immunogenecity).
2 5 Monoclonal antibodies to proteins derived from CMV are useful tools for
the
detection of CMV expression in cells and cell extracts both in vivo and in
vitro, for
purification purposes and for a variety of biochemical and immunological
analysis
techniques to study the function of these proteins.
3 0 The term "immunochemically reagent" according to the invention usually
consists
of one or more (monoclonal) antibodies and a suitable support or a labeling
substance.
Supports which can be used are, for example, the inner wall of a microtest
well or
a cuvette, a tube or capillary, a membrane, filter, test strip or the surface
of a particle such
as, for example, a latex particle, an aldehyde particle (such as a ceramic
magnetizable
35 particle with active aldehyde surface groups), an erythrocyte, a dye sol, a
metal sol or
metal compound as sol particle, a carrier protein such as BSA or KLH.
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Labeling substances which can be used are, inter alia, a radioactive isotope,
a
fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol
particle.
The invention further comprises the use of antibodies to said peptide in
5 immunological and biochemical methods aiming to detect the full length
protein in a test
fluid or tissue specimen.
Antibodies, both monoclonal and poIyclonal, directed against peptides
according to
the invention are very suitable in diagnosis and immunocytochemistry for
detection in situ
2 0 in tissue specimen, while those antibodies which are neutralizing are very
useful in passive
immunotherapy.
Part of the invention is also the "humanizing" of the monoclonal antibodies in
question. Techniques for raising the "humanized" monoclonal antibodies are
known in the
15 art.
For the detection of CMV in a sample a peptide reagent according to the
invention,
containing one or more peptides, can be brought into contact with the sample
and anti
CMV after which the presence of immune complexes formed can be detected and,
from
2 0 this, the presence of CMV in a sample can be determined.
A particularly suitable method for the detection of CMV in a sample is based
on a
competition reaction between a peptide used in said peptide reagent provided
with a
labeling substance and a CMV antigen (present in the sample) whereby the
peptide and the
2 5 antigen are competing with the antibody directed against CMV attached to a
solid support.
The invention further comprises a method for the detection of cytomegalovirus
{CMV) in a sample characterized in that an antibody according to the invention
is brought
into contact with a sample after which the presence of immune complexes formed
is
30 detected which is a measure for the presence of CMV in the sample.
A test kit according to the invention comprises as an essential constituent a
peptide
reagent as described above. Carrying out a sandwich reaction, for the
detection of CMV
antibodies the test kit may comprise, for example, a peptide used in a peptide
reagent
according to the invention coated to a solid support, for example the inner
wall of a
35 microtest well, and either a labeled peptide used in a peptide reagent
according to the
invention or a labeled anti-antibody.
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For carrying out a competition reaction, the test kit may comprise a peptide
used in
a peptide reagent according to the invention coated to a solid support, and a
labeled
antibody directed against CMV preferably a monoclonal antibody directed
against said
peptide.
In an agglutination reaction the test kit comprises a peptide reagent
comprising a
peptide coated to particles or sols.
Another embodiment of a test kit is, for example, the use of a labeled peptide
used
in a peptide reagent according to the present invention in a competition
reaction with a
CMV antigen to be detected for a binding site on the antibody directed against
CMV,
which is coated to a solid support.
The above disclosure generally describes the present invention. A more
complete
understanding can be obtained by reference to the following specific examples
which are
provided for purposes of illustration only, and are not intended to limit the
scope of the
2 5 invention.
Brief description of the figures:
Peptide code definition:
#K-38-E {pp150): SEQ.ID.No.
1
#T-21-C (pp150): SEQ.ID.No.2
#C-21-M (pp150): SEQ.ID.No.3
#T21CC21M (pp150): SEQ.ID.No.4
#OTP194(pp150): SEQ.ID.No.S
#OTP197(pp150): SEQ.ID.No.6
#OTP101A (gB): SEQ.ID.No.7
#F-28-G (pp52): SEQ.ID.No.8
#OTP118A(pp28): SEQ.ID.No.9
#OTP 119A (pp28): SEQ.ID.
No.10
Figure 1:
Identification of binding sites for human IgG antibodies (10 sera of CMV
seropositive individuals) on CMV-pp28(UL99) by peptide-scanning ELISA:
A = the relative absorbance values for each individual serum tested
B= the sum of individual absorbance values for each peptide, corrected for the
background value for each serum
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C= the positivity-score for each peptide yielding an absorbence value of at
least 3
SD above the mean background value for each serum tested.
Figure 2:
Analyses representing the best set of peptides reacting with IgG antibodies
using
sera from patients with active CMV-infections and appropriate controls without
active
CMV-infections.
Figure 3:
Analyses representing the best set of peptides reacting with IgM antibodies
using
sera from random healthy individuals seropositive or seronegative for CMV
antibodies as
determined by reference antibody assays.
Figure 4:
Comparison of the reactivity of human IgG with cell culture derived CMV-Ag
(horizontal axis) versus a CMV peptide reagent [#K38E + #OTP101A + #OTP119A]
(SEQ.ID.No. 1 + SEQ.ID.No. 7 + SEQ.ID.No. 9).
Figure 5:
A comparison of the reactivity of human serum IgM is shown with cell culture
derived CMV-Ag (horizontal axis) versus a CMV peptide reagent [#K38E + #F28G]
(SEQ.ID.No. 1 + SEQ.ID.No. 8) on the vertical axis.
Figure 6:
Immunoblot detection of CMV-pp150(UL32) using CMV.OT3C. Control reactions
with monoclonal antibodies to additional CMV proteins (GICR series obtained
from
Goodwin Institute for Cancer Research, Florida, USA) and human serum IgG from
a
CMV-seropositive donor are shown in the parallel strips as indicated in the
legend below
the figure.
The invention is further exemplified by the following examples:
Example 1.
Peptides with a length of 12 amino acids (AA) and overlapping by 11 AA of the
AA sequence of the complete UL99 reading frame reading frame encoding CMV-pp28
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were synthesized by automated solid phase synthesis onto chemically activated
pins as
originally described by Geysen et al (P.N.A.S.,USA,83 {1984) p.3998-4002).
The immunoreactivity of each peptide with CMV-specific antibodies from human
sera ( 10 CMV seropositive individuals) was determined as described by
Middeldorp and
Meloen (J.Virol.Meth.21 (1988) p.147-159).
The result of such a PEPSCAN analysis with 10 sera from different CMV-
seropositive individuals is shown in Figure 1.
Panel A shows the relative absorbance values for each individual serum tested.
Panel B shows the sum of individual absorbance values for each peptide,
corrected
for the background value for each serum.
Panel C shows the positivity-score for each peptide yielding an absorbence
value of
at least 3 SD above the mean background value for each serum tested.
The background absorbance value for each serum was determined as the mean
absorbance value of the 10 least reactive peptides.
From this figure it can be seen that most sera contain antibodies reactive
with
distinct regions of the pp28 amino acid sequence. Some regions are recognized
by multiple
individuals, thus representing immunodominant epitopes of pp28.
Similar date have been obtained by PEPSCAN analysis of other CMV proteins such
as pp150(UL32), pp65{UL83), pp52(UL44) .
Conclusion: Antibodies in the sera from different individuals recognize CMV
proteins through interaction with distinct regions (epitope clusters), the
position of which
2 5 may differ significantly for each individual.
Example 2.
Selected peptides of different length (see sequence listing) were synthesized
using
3 0 standard solid phase synthesis to combine multiple PEPSCAN reactive
domains into a
single molecule.
These peptides were coated onto the solid phase in the wells of 96-well micro-
ELISA plates, usually at l~,g/ml in coating buffer and non-bound positions
were blocked
with 1 % bovine serum albumine (BSA) in coating buffer. Peptides were coated
directly or
3 5 as BSA-peptide complexes created by glutaraldehyde mediated coupling of
peptides to
highly purified BSA, depending to the best immunochemical reactivity as
determined in
optimization experiments.
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After coating, the wells were washed with 0.1 M phosphate buffered saline
pH7.4
(PBS) containing 0.05°b Tweeri 20 (PBS-T) and dilutions of human sera
in PBS-T (usually
1:100) were analyzed for antibody reactivity using standard ELISA procedures.
In figure 2 the peptide ELISA results are shown for IgG antibodies using sera
from
random healthy individuals seropositive or seronegative for CMV antibodies.as
determined
by reference antibody assays.
In the following table, for each panel is the corresponding peptide analysis
is
shown:
l0 Panel 2A - peptide ~I21C (SEQ.ID.No. 2),
Panel 2B - peptide ~CZ I M (SEQ.ID.No. 3),
Panel 2C - peptide #T21 CCZ 1 M (SEQ.ID. No. 4),
Panel 2D - peptide #K3$E (SEQ.ID.No. 1),
Panel 2E - peptidev4fOTP194 (SEQ.ID.No. 5),
Panel 2F - peptide ~IOTPI97 (SEQ.ID.No. 6),
Panel 2G - peptide ~F28G (SEQ.ID.No. 8),
Panel 2H - peptide ~OTP IOIA (SEQ.ID.No. 'n,
Panel 21 - peptide #OTP 118A (SEQ.ID. No: 9),
Panel ZJ - peptide ~OTP I 19A (SEQ.ID. No. I0),
Panel 2K - CMV-Ag being the cell culture extract (golden standard).
In each panel 4 such experiments are shown with different sets of human sera
obtained from random healthy blood donors.
The CMV immune status was determined using a reference "golden standard" (*)
ELISA based upon cell culture derived CMV antigens (Panel 2K).
(o) represents CMV seropositive samples
(x) represents CMV seronegative samples.
(*) Antigens for the "golden standard" consisted of purified de-enveloped
virions
and dense bodies, prepared from human embryonic lung fibroblasts, infected for
6 days
with 3PFU/cell of CMV strain AD-169. Infected cells were freeze-thawed _3
times and
extracted with 0.35°6 TX100*~in O.OSM phosphate buffer pH7.4 to release
intra~oeliular
virions and dense bodies into the soluble phase. Soluble particles were
separated from
nuclei and larger cellular material by density centrifugation over a Ficoll*
layer for 20 min.
at 1200xg at +4°C. From the upper layer viral particles were isolated
by centrifugation at
12.OOOxg for 10 minutes and the pelleted particles were solubilised by short
sonication in
PBS. By election microscopy the particles were shown to consist predominantly
of de=
enveloped viral capsids and dense bodies:
*Trade-mark
CA 02259967 1999-O1-11
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In figure 3 a selection of the results is shown of analyses representing the
best set of
peptides reacting with IgM antibodies using sera from patients with active CMV-
infections
and appropriate controls without active CMV-infections.
5 Sera were obtained from renal transplant patients with active CMV
infections,
showing a positive reaction for CMV-IgM in the reference ELISA (See Fig.3E).
In the following table, for each panel is the corresponding peptide analysis
is
shown:
Panel 3A - peptide #K38E (SEQ.ID.No. 1),
10 Panel 3B - peptide #T21CC21M (SEQ.ID.No. 4),
Panel 3C - peptide #F28G (SEQ.ID.No. 8),
Panel 3D - peptide #OTP119A (SEQ.ID.No. 10),
Panel 3E - CMV-Ag being the cell culture extract (golden standard).
15 Conclusion: The data shown in figures 2 and 3 indicate that individual
combi-
peptides are reactive with antibodies in most human sera but that no peptide
shows
reactivity with all sera. Occasionally some peptides react (false-positive)
with CMV-
negative sera.
Therefore, although most selected peptides appear to be reactive with several
2 o human sera no single peptide is reactive with all sera.
Example 3.
In several experiments performed it was found that selected combinations) of
well
defined synthetic combi-peptides derived from different immunoreactive CMV-
proteins,
especially the combination of [#K38E + #OTP101A + #OTP119A]( SEQ.ID.No. 1 +
SEQ.ID.No. 7+ SEQ.ID.No. 10), were capable of detecting CMV-IgG antibodies in
human sera with equal or better sensitivity than current "golden standard"
ELISA using
CMV antigens derived from cell culture.
CMV-Ag was prepared and coated on the solid phase as described in Example 2.
#OTP101A (SEQ.ID.No. 7) was coupled to BSA before coating in equimolar ratio
with
#K38E (SEQ.ID.No. 1) and #OTP119A (SEQ.ID.No. 10).
In Figure 4 data is shown wherein CMV-IgG reactivity in a peptide combination
ELISA is compared with the reference ELISA which uses semi-purified cell
culture
derived CMV antigen on the solid phase. The sera analysed were from healthy
blood
3 5 donors either from the USA (4A) or from The Netherlands (4B).
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The CMV-IgG reactivity as determined in the peptide-based ELISA (vertical
axis)
gives equal or even better reactivities compared to the "golden standard"
ELISA
(horizontal axis). Control human sera, negative for CMV-IgG are negative in
both assays.
The presence of all three peptides on the solid phase was checked using
epitope-
S specific antibodies (data not shown).
Conclusion: The availability of such a well specified set of CMV synthetic
molecules is of value for the development of highly defined assays for the
detection of anti
CMV IgG in human sera and may contribute to further standardization of CMV
1 o serodiagnosis and determination of CMV-immune status.
Example 4.
Combination of multiple selected combi-peptides in a single well was also
necessary
to obtain sufficient reactivity with human anti-CMV IgM as no single peptide
was capable
15 of reacting to a similar extend as cell culture CMV-Ag with all sera that
were analysed
{Example 2).
Therefore several combinations of peptides were tested and optimized.
From these results the best combination of peptides for the detection of human
serum anti-CMV IgM proved to be [#K38E + #F28G] (SEQ.ID.No. 1 + SEQ.ID.No. 8).
20 Figure 5 shows the comparison of the reactivity of human serum IgM with
cell
culture derived CMV-Ag and [#K38E + #F28G] (SEQ.ID.No. l + SEQ.ID.No. 8) bound
to the solid phase at l~,g/mt.
Sera were obtained from renal transplant patients with a well defined primary
CMV-infection.
25 CMV-Ag were prepared and coated as described in Example 2 and peptides were
coated simultaneously onto the solid phase each at I,uglml in O.OSM carbonate
buffer
pH9.6.
It can be concluded that [#K38E + #F28G) (SEQ.ID.No. 1 + SEQ.ID.No. 8)
provides an excellent and well defined combination capable of replacing cell
culture
30 derived CMV-Ag in the detection of human anti-CMV-IgM antibodies.
Example 5.
Monoclonal antibodies reactive with CMV-peptide reagents according to the
present
invention are useful for diagnostic assays (e.g. as CMV-specific conjugate in
IgM-capture
35 assays, as quality control to determine peptide coating on solid phase).
Such antibodies may also be useful in the direct detection of CMV in cultured
cells
and patient tissue or body fluids.
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22
For illustration only one of such an antibody, named CMV.OT3C (deposited at
the
European Collection of Animal Cell Cultures, CAMR (Cenue for Applied
Microbiology &
Research), Salisbury (UK) under deposit No. 96071123) directed against the C-
terminal
domain of CMV-pp150 (UL32) and reactive with peptide #K38E (SEQ.ID.No. 1) by
ELISA, is described. The antibody is capable of detecting intact ppISU in CMV-
infected
cell lysates by ELISA, immunoblot or immunoprecipitation and specifically
stains CMV-
infected cells by immunofluorescence or immunohistochemical techniques.
Direct enzyme coupling of CMV.OT3C yields an antibody conjugate which may be
1 o employed as CMV-specific detector reagent in (IgM or IgA) immunocapture
assays.
In figure 6, an immunoblot is shown which was prepared as follow: Prot~ns~ .of
CMV-AD169 infected human fibroblast whole cell lysate were denatured and
separated by
standard Laemmli SDS-P~4GE and blotted, onto nitrocellulose. Strips were cut
and
remaining protein binding sites were blocked by incubation for one hour with
496 dry milk
powder dissolved in PBS with 5 ~& horse serum(blocking buffer). Subsequently
the strips
were incubated with monoclonal antibody in blocking buffer for one hour at
37°C. After
washing with PBS-Tweeri anti-mouse HRP conjugate was added and incubated as
above
aid bound enzyme reactivity was detected using 4-chloronaphtol as
precipitating substrate.
2 0 Control reactions with monoclonal antibodies to additional CMV proteins
(GICR
. series obtained from Goodwin Institute for Cancer Research, Florida, USA)
and human
serum IgG from a CMV-secopositive donor are shown in the parallel strips as
indicated in
the legend below the figure.
*Trade-mark
CA 02259967 1999-07-07
23
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: AKZO NOBEL N.V.
(ii) TITLE OF INVENTION: PEPTIDE REAGENT FOR THE DETECTION
OF HUMAN CYTOMEGALOVIRUS (CMV)
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FETHERSTONHAUGH & C0.
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
2 0 (D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,25!3,967
(B) FILING DATE: 09-JUL-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 96201972.5
(B) FILING DATE: 12-JUL-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FETHERSTONHAUGH & CO.
3O (B) REGISTRATION NUMBER:
23804-529
CA 02259967 1999-07-07
23a
(C) REFERENCE/DOCKET NUMBER: 23804-529
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-235-4373
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
23804-529
CA 02259967 1999-O1-11
WO 98/02746 PCTIEP97/03717
24
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
Lys Ser Gly Thr Gly Pro Gln Pro Gly Ser Ala Gly Met Gly Gly Ala
1 5 10 25
Lys Thr Pro Ser Asp Ala Val Gln Asn Ile Leu Gln Lys Ile Glu Lys
20 25 30
Ile Lys Asn Thr Glu Glu
15 (2} INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
20 (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Thr Pro Thr Pro Val Asn Pro Ser Thr Ala Pro Ala Pro Ala Pro Thr
5 10 15
35 Pro Thr Phe Ala Cys
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{2) INFORMATION FOR SEQ ID NO: 3:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
5 (B) TYPE: amino acid
{C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A} ORGANISM: Cytomegalovirus
(xi} SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Cys Gln Thr Pro Val Asn Gly Asn Ser Pro Trp Ala Pro Thr Ala Pro
1 5 10 15
Leu Pro Gly Asp Met
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
CA 02259967 1999-O1-11
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26
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Thr Pro Thr Pro Val Asn Pro Ser Thr Ala Pro Ala Pro Ala Pro Thr
1 5 10 15
Pro Thr Phe Ala Cys Cys Gln Thr Pro Val Asn Gly Asn Ser Pro Trp
20 25 30
Ala Pro Thr Ala Pro Leu Pro Gly Asp Met
35 40
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
Thr Asp Thr Glu Thr Ser Ala Lys Fro Pro Val Thr Thr Ala Tyr Lys
1 5 10 15
Phe Glu G1n Pro Thr Leu Thr Phe Gly Ala Gly Val Asn
20 25
(2) INFORMATION FOR SEQ ID NO: 6:
CA 02259967 1999-O1-11
WO 95102746 PCT/EP97103717
27
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Asp Gly Tyr Pro Pro Asn Arg Gln Asp Pro Arg Phe Thr Asp Thr Leu
1 5 10 15
Val Asp Ile Thr Asp Thr Glu Thr Ser Ala Lys
20 25
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
EA) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CA 02259967 1999-O1-11
WO 98/02746 ~ PCTIEP97J03717
28
Ser Glu Ala Val Ser His Arg Ala Asn Glu Thr Ile Tyr Asn Thr Thr
1 5 10 15
Leu Lys Tyr Gly Asp Val
(2) INFORMATION FOR SEQ ID NO: 8:
10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Phe Leu Thr Glu Glu Pro Phe Gln Arg Gly Asp Pro Phe Asp Lys Asn
1 5 10 15
Tyr Val Gly Asn Ser Gly Lys Ser Arg Gly Gly Gly
20 25
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02259967 1999-O1-11
WO 98/02746 PCT/EP971037I7
29
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Thr Thr Pro Gly Glu Pro Leu Lys Asp Ala Leu Gly Arg Gln Val Ser
1 5 10 15
Leu Arg Ser Tyr Asp Asn Ile Pro Pro Thr Ser Ser Ser Asp Glu Gly
20 25 30
Glu Asp Asp Asp Cys
20 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
2~ (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
30 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Cytomegalovirus
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Cys Glu Thr Asp Asp Leu Asp Glu Glu Asp Thr Ser Ile Tyr Leu Ser
CA 02259967 1999-O1-11
WO 98/02746 PCT/EP97/03717
1 5 10 15
Pro Pro Pro Val Pro Pro Val Gln Val Val Ala Lys Arg Leu Pro Arg
20 25 30
5
Pro Asp Thr Pro Arg Thr
