Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND KIT FOR DETERMINING SEVERE INFLAMMATORY
REACTIONS TO MOSQUITO BITES
CROSSREFERENCE TO RELATED APPLICATIONS
This application is a conversion of a Provisional
Application filed November 13, 1997, Serial No.
60/065,402.
BACKGROUND OF THE INVENTION
Mosquito bites are a global problem not only
because they facilitate transmission of potentially
fatal diseases such as malaria and yellow fever, but
also because they cause local skin reactions and,
rarely, systemic reactions including urticaria,
angioedema, and even anaphylactic shock [Frazier, 1973;
McCormack et al., 1995]. Skin reactions to mosquito
bites are caused by the proteins in the mosquito saliva
that enter the skin when mosquitoes take a blood meal
[Hudson et al., 1960]. Mosquito saliva proteins elicit
both IgE-mediated immediate hypersensitivity and
lymphocyte-mediated delayed hypersensitivity [Oka 1989;
Peng et al., 1996]. Mosquito salivary proteins are also
involved with many aspects of the process of
hematophagy which provide new perspectives for
evaluating the transmission dynamics of pathogens
(James, 1994] .
Mosquito saliva contains a complex of proteins.
Protein visualization techniques using gel
electrophoresis and silver staining have revealed as
many as 20 peptides in adult mosquito Aedes aegypti
saliva [Racioppi and Spielman, 1987], which include cx-
amylase, anticoagulants, anti-TNF, apyrase, esterase,
D7, a-glucosidase, and sialokinins [James, 1994].
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Immunotherapy is successful in the treatment of
mosquito allergy [Frazier, 1973; Gluck and Pacin, 1968;
McCormack, 1995; Tager et al, 1969; Benaim-Pinto and
Fassrainer, 1990], correlating with the observation
that natural desensitization eventually occurs during
long-term exposure to the bites [Mellanby, 1946; McKiel
and West, 1961; Peng, et al, 1994, 1996]. This therapy
is neither well studied nor widely used at least in
part because commercially available mosquito extracts
have never been standardized and contain many non-
saliva proteins which may cause sub optimal efficacy
and side effects.
Commercial extracts of mosquito saliva protein are
available. Commercial mosquito extracts contain
multiple proteins and antigens since they are whole
body extracts (Example 1). Many of the antigens in
these extracts are unrelated to the allergens in
mosquito saliva. The antibodies exhibited in patients
which are directed against these non-saliva antigens in
the commercial extracts may have been induced by
inhalation of insect particles or by being bitten by
other insects whose antigens cross-reacted with
mosquito body components leading to the formation of
IgE and IgG antibodies against mosquito body antigens.
Commercial mosquito extracts should be standardized
given their extreme heterogeneity (Example 1) and purer
mosquito extracts should be used in the diagnosis and
immunotherapy of mosquito allergy. In addition to the
poor diagnostic accuracy of skin testing, injection of
crude extract may lead to the development of
sensitization to some of its components [Hamilton,
1990]. It has further been demonstrated that the
efficacy of immunotherapy is considerably improved by
the use of purified antigens [Swan, 1989].
Purification or isolation of each of the saliva
proteins is required to improve studies of the
diagnosis and immunotherapy of mosquito allergy, and --
studies of mechanisms of mosquito-transmitted diseases.
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However, purification of each salivary protein from
mosquito whole body extract is an extremely laborious
task and may result in the potential loss of important
allergens or their biologic activity during the
necessary multiple purification procedures. Collection
of mosquito saliva is time-consuming and labor-
intensive and therefore is also impractical.
Utilization of molecular techniques to clone and
express masquito salivary proteins would be useful in
developing allergens for skin testing and
immunotherapy. However, not all recombinant proteins
can be used as allergens since when the proteins are
expressed glycosylation patterns of the proteins are
not always followed as well as other folding parameters
so that it is not clear that recombinant proteins will
elicit the correct immunological response including
biological activity.
To date, two salivary gland cDNAs of Aedes aegypti
which encode a 68 kDa protein (apyrase) and a 37 kDa
one (D7) have been cloned [James et al., 1991; Smartt
et al., 1995]. However, these proteins need to be
characterized to determine if they are allergens, if
they are shared by other mosquito species and whether
they would be useful in a standardized extract for the
use in skin tests and immunotherapy.
To facilitate studies of mosquito allergy
additional recombinant proteins are needed that are not
species-shared and which can be used as an allergen in
a standardized extract for skin testing and
immunotherapy (desensitization). In addition,
recombinant proteins that are species-specific are also
needed so that appropriate combinations of allergens
can be made that are patient-specific.
SiII~SARY OF THE INVENTION
According to the present invention, a recombinant
mosquito salivary allergen for use in skin tests,
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immunoassays and immunotherapy for allergic reactions
to mosquito bites is provided. The recombinant
allergen is produced by a cDNA encoding an IgE-binding
protein or fragment thereof or analogue thereof which
is found in mosquito saliva. In an embodiment the
recombinant allergen is rAed a 1, a 68 kDa recombinant
allergen, or rAed a 2, a 37 kDa recombinant allergen or
rAed a 3, a 30 kDa recombinant allergen. The
recombinant allergen may share common allergenicity
among at least two species of mosquitos or may be
species-specific.
The present invention also provides a method of
skin testing and undertaking immunotherapy utilizing
the recombinant salivary allergen and a kit for
practicing the method of the invention. The
recombinant allergens are selected to share common
allergenicity among the mosquito species for which
testing or treatment is required. In a preferred
embodiment a combination of allergens with common
specificities among species is used so as to
effectively represent the mosquito species distribution
of a wide geographic area for which testing and/or
immunotherapy is needed.
The present invention also provides an immunoassay
for measurement of mosquito salivary allergen-specific
IgE and IgG using recombinant mosquito salivary
allergen as the substrate to which the allergen-
specific IgE and IgG binds. The present invention also
provides a kit for the immunoassay including the
appropriate recombinant allergen, antibody directed to
the allergen and may also contain reference sera.
The present invention further provides antibodies
directed against the recombinant salivary allergens,
wherein the recombinant allergen is produced by a cDNA
encoding an IgE-binding protein or fragment thereof or
analogue thereof which is found in mosquito saliva. In
an embodiment, the antibody is directed against rAed a--
1, a 68 kDa recombinant allergen, or rAed a 2, a 37 kDa
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recombinant allergen or rAed a 3, a 30 kDa recombinant
allergen. The antibody may be polyclonal or
monoclonal. The antibodies are used for immunoassays,
purification and antigen standardization.
DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention will be
readily appreciated as the same becomes better
understood by reference to the following detailed
description when considered in connection with the
accompanying drawings wherein:
Figure 1 shows the results of using nitrocellulose
filters to characterize cDNA coding protein using
specific mouse and human antibodies to the protein;
Figure 2 shows results for mean mosquito-specific
IgE levels of three species and the subjects with or
without immediate skin reaction to mosquito bites;
Figure 3 shows the IgE (left) and IgG (right)
responses to the antigens of Ae. vexans in three
subjects with severe skin reactions to the bites (strip
number 1-3) and two subjects without skin reactions to
the bites (strips number 4 and 5);
Figure 4 shows a comparison of IgE and IgG
responses in three subjects;
Figure 5 shows the results of skin epicutaneous
tests;
Figure 6 shows the results of testing of nine
subjects with positive rAed a 1 reactions;
Figure 7 shows Western blot analysis showing
inhibition of binding by addition of mosquito head and
thorax extract in a dose-dependent manner;
Figure 8 shows Aed a 2 to be a species-shared
allergen being present in saliva or salivary glad
extracts;
Figure 9 shows individual IgE responses to
allergens evaluated in twelve mosquito-allergic --
subjects living in Canada, USA and China as well as
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five subjects who were not allergic to mosquito bites,
immunoblotting being performed using two rabbit
antibodies specific to recombinant mosquito salivary
proteins; and
Figure 10 is an immunoblot using rabbit anti-Aed a
1 and Aed a 2 antibodies respectively.
DETAILED DESCRIPTION OF T8E PREFERRED EMBODIMENT
The present invention provides recombinant
mosquito salivary allergens for use in skin tests,
immunoassays and immunotherapy. The recombinant
allergen is identified and produced by a cDNA encoding
an IgE-binding protein or fragment thereof or analogue
thereof which is found in mosquito saliva as is shown
in the Examples herein.
The allergen is generally a protein or protein
fragment or analogue thereof found in the saliva of
mosquitos as described herein above. The allergen
elicits an IgE response and may also elicit an IgG
response. The fragment contains some or all of the
epitopes on the whole allergen. It elicits an IgE
response and may also elicit an IgG response. The
recombinant allergen expresses the same epitopes,
either sequence based or conformational, as the native
allergen and shares the same antigenic functions as the
native allergen. The antigenic functions essentially
mean the possession of an epitope or antigenic site
that is capable of cross-reacting with antibodies
raised against a naturally occurring salivary protein.
Further, the recombinant antigen must also have the
same biologic activity, that is it must elicit an
immune response in vi vo.
The term "analogue" as used herein is defined as a
variant (alternatively the terms alteration, amino acid
sequence alteration, amino acid sequence variant can be
used) with some differences in their amino acid -
sequences as compared to the native sequence of
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salivary proteins, but functionally equivalent.
Ordinarily, the analogue will be generally at least 70%
homologous over any portion that is functionally
relevant for eliciting an immune response. In more
preferred embodiments the homology will be at least 80%
and can approach 95% homology to the mosquito salivary
protein sequence. The homology will extend over a
region of at least nine contiguous amino acids. The
amino acid sequence of an analog may differ from that
of the native protein when at least one residue is
deleted, inserted or substituted, but the protein
retains its antigenic competence and biological
activity in vivo in relation~to eliciting an immune
response. Differences in glycosylation can provide
analogues.
Functionally equivalent refers to the biological
property of the molecule and in this context means an
in vivo eliciting of an immune response by a naturally
occurring (native) salivary proteins. The antigenic
functions essentially mean the possession of an epitope
or antigenic site that is capable of cross-reacting
with antibodies raised against a naturally occurring
salivary protein and eliciting skin reactions as do the
native salivary allergens. Biologically active (in
vivo activity) means that the analogues share an
antigenic function and elicit an immune response in
vi vo .
Any expression system may be used as is known in
the art that will provide recombinant allergens or
allergen fragments that express the eliciting epitopes
and have activity in vivo. In a preferred embodiment
the allergen is produced in a baculovirus expression
vector system.
As exemplars, recombinant allergens rAed a 1, a 68
kDa recombinant allergen, and/or rAed a 2, a 37 kDa
recombinant allergen and/or rAed a 3 (SEQ ID No:l), a __
30 kDa are used. The allergens are named using the
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rules as set forth in Larson and Lowenstein [1996}.
The recombinant allergens can be selected such
that they share common allergenicity among at least two
species of mosquitos as shown in the Examples.
Alternatively, the allergen is species specific.
The allergens used for skin testing are selected
on the basis of the mosquito species distribution in
the geographic area in which the patient to be tested
is exposed or will be exposed. It is contemplated by
the present invention that in one embodiment patients
moving into new geographic areas or planning to
vacation in a new geographic area will be skin tested
to determine their sensitivity to the predominant
mosquito species population of that area and can then
be desensitized as described herein below.
As shown in the Examples herein below the
allergens may be cross-reactive across several species
or may be species-specific. The skin testing will
utilize the recombinant allergens that effectively
represent the mosquito species distribution for the
geographic area in which the patient is exposed or will
be exposed.
The concentration of each recombinant allergen
used in skin testing will be determined using in vivo
and/or in vitro standardization techniques. The
standardization techniques have been described in
detail previously [Ipsen et al. 1993} and are
summarized herein below. Each recombinant allergen
extract will be standardized against a standard
reference to assure lot-to-lot consistency and relative
potency of allergenic recombinant extracts.
In addition to, or alternatively, to the skin
testing the response to the recombinant mosquito
allergens can be determined by an immunoassay as is
known in the art and described herein below. In
general an ELISA for IgE is preferred but Western
Blotting can also be used. The present invention also
provides a kit including the appropriate recombinant
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allergen, antibody directed to the allergen and may
contain reference sera for the practice of the
immunoassay.
The present invention also provides an allergy
immunotherapy medicament which contains the recombinant
mosquito allergens for use in immunotherapy
(desensitization) as described herein below. The
recombinant allergens are suspended in pharmaceutically
acceptable carriers, diluents, adjuvants and/or
vehicles as is known in the art of immunotherapy.
These pharmaceutically acceptable carriers and the like
are selected such that they do not react with the
active ingredients of the invention and that the
allergens retain their immunologically eliciting
conformation and biological activity.
It should be noted that an alternative term for
the immunotherapy medicament is "extract" which is
generally used to indicate allergens (immunogens) that
have been isolated or prepared from a native or natural
source and not produced recombinantly. An example of
an extract would be mosquito whole body preparations.
However, the allergy immunotherapy medicament of the
present invention can be referred to as a "recombinant
extract".
The allergy immunotherapy medicament will contain
at least one recombinant mosquito allergen. In
general, the recombinant mosquito allergens are
selected on the basis of the skin test results. The
starting dose of the allergen can be determined by skin
test endpoint-titration using a dose that is equal to
0.1 ml of the end-point dilution that initiates a skin
reaction or other methods known in the art. The
medicament will contain a combination of recombinant
allergens that effectively represent the mosquito
species distribution for the geographic area to which
the patient is allergic and for which they need
desensitization. --
The present invention provides a kit for skin
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testing for allergy to mosquito bites and a kit for
providing immunotherapy including recombinant mosquito
salivary allergens. In general the kit includes
recombinant allergens that are selected to share common
allergenicity among the mosquito species common to the
geographic area for which testing or immunotherapy is
required. The kit may also include species-specific
recombinant allergens for each mosquito species common
to the geographic area for which testing and/or
immunotherapy is required. In an embodiment, the kit
includes recombinant allergens rAed al, a 68 kDa
recombinant allergen, rAed a2, a 37 kDa recombinant
allergen and rAed a3, a 30 kDa recombinant allergen.
Since 1865, skin tests have been used to provide
helpful confirmatory evidence for diagnosis of specific
allergy. Skin tests include epicutaneous and
intradermal tests. Detailed techniques have been
previously described (Bousquet and Michel 1993].
Briefly, in the epicutaneous tests, drops of
recombinant allergen extracts are placed approximately
2 cm apart on the volar surface of the forearm. The
point of a disposable needle is passed through the
drop, inserted into the epidermal surface, and then
gently lifted without inducing bleeding. The immediate
wheal and flare reactions are traced and recorded 20
minutes after the teat, and delayed indurated papules
are traced 24 hours later. In the intradermal tests, a
volume of approximately 0.01 to 0.05 ml of the
recombinant allergen is injected into the akin to
produce a small superficial bleb approximately 2-3 mm
in diameter. Results are traced and recorded in the
same manner as for the epicutaneous tests.
Since 1911, immunotherapy injections with
increasing amounts of the offending specific
allergen(s), have been successfully used to relive
allergic symptoms after subsequent exposure to the
allergen(s). This therapy is still one of the major --
therapeutic methods for treatment of patients with
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allergic diseases. The method is described in detail
by TE van Metre and NFA Adkinson, Jr., [1993]. First,
the recombinant mosquito allergens to which the
subjects are allergic are determined by skin tests
and/or measurement of specific IgE. These recombinant
allergens are then combined and used for the
immunotherapy. Subcutaneous injections of the
allergens will be given weekly or twice a week in
gradually increasing doses. A starting dose will
contain 0.5 allergy units/ml or can be chosen by skin
test end-point titration using a dose that is equal to
0.1 ml of the end-point dilution that initiate skin
reaction. When the highest tolerated dose is reached,
this dose is used to maintain allergen-specific
immunity, that is, injected every 2 - 4 weeks for a
period of time, as determined by clinical history and
monitored immunologic tests.
Currently, native allergens (proteins) are used in
immunotherapy. Recent studies have shown that
immunotherapy with the genes (plasmid DNA) which encode
for allergen inhibit specific IgE response, histamine
release, and allergen-induced airway
hyperresponsiveness [Hsu et al. 1996; Raz et al. 1996].
Therefore, immunotherapy with plasmid DNAs encoding or
mosquito salivary allergens is contemplated by the
present invention for immunotherapy for people who are
highly reactive to mosquito bites.
Diagnosis of mosquito allergy can be also made by
measurement of serum recombinant allergen-specific IgE
and IgG antibodies using immunoassays. In general,
ELISAs are the preferred immunoassays employed to
assess the amount of IgE and IgG in a specimen. ELISA
assays are well known to those skilled in the art.
Both polyclonal and monoclonal antibodies can be used
in the assays. Where appropriate, other immunoassays,
such as radioimmunoassays (RIA) can be used as are
known to those in the art. Available immunoassays are
extensively described in the patent and scientific
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literature. See, for example, United States patents
3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;
3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771
and 5,281,521.
Any expression system as is known in the art that
will provide recombinant allergens or allergen
fragments that express the eliciting epitopes and have
activity in vivo can be used in the practice of the
present invention. In a preferred embodiment the
baculovirus insect cell expression system, which
performs many of the post-translational modifications
found in mammalian cells, is an excellent system for
the production of large amounts of biologically active
proteins (see generally O'Reilly et al, 1994.
Baculovirus Expression Vectors: A Laboratory Manual.
Oxford University Press). The efficiency of expression
of baculovirus system differs from gene to gene by
approximately 1000-fold. In particular this system
provides for glycosylation such that proper
carbohydrate expression is provided which plays a role
in the immunological response and in vivo activity.
Vectors can be constructed containing the cDNA of
the present invention, by those skilled in the art, and
should contain all expression elements necessary to
achieve the desired transcription of the sequences in
the selected expression system. Other beneficial
characteristics can also be contained within the
vectors such as mechanisms for recovery of the nucleic
acids in a different form. The vectors can also
contain elements for use in either procaryotic or
eucaryotic host systems. One of ordinary skill in the
art will know which host systems are compatible with a
particular vector.
The vectors can be introduced into cells or
tissues by any one of a variety of known methods within
the art (calcium phosphate transfection;
electroporation; lipofection; protoplast fusion;
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polybrene transfection). The host cell can be any
eucaryotic and procaryotic cells, which can be
transformed with the vector and which will support the
production of the allergen with proper glycosylatian
and conformation.
pBlueBacHis vectors are designed for efficient
expression and purification of recombinant proteins
[Chen et al, 1993; Reddy et al, 1994; Rotrosen et al,
1993; O'Reilly et al, 1994; Matsuura et al., 1987; Rupp
et al., 1995; Chalkley et al, 1994] and have been used
by applicants. Since the variety of expression vector
may also play a role in the expression levels of
baculovirus system, expression vectors are selected for
each recombinant allergen as is known iri the art such
that the yield of allergen is maximized.
Expressed recombinant allergens in the cell
culture media will be purified according to the physio-
biochemical characteristics of each recombinant
allergen as is known in the art. For example,
histidine tagging fXu et al., 1996] for purification of
recombinant proteins by immobilized metal affinity
chromatography can be used for a number of proteins
both in prokaryotic [Dudler et al., 1992.] and
eukaryotic (Janssen et al., 1995; Reddy et al, 1994]
expression systems. Other purification systems as are
known in the art can be used including affinity
chromatography utilizing the monoclonal antibodies to
recombinant allergens of the present invention.
Alternatively, a combination of ion exchange (DEAE
Sephacel) and gel filtration (Sepgacryl S-100)
chromatograph can be used. Whatever method of
purification that is used will be selected such that
the isolated and purified recombinant allergen will
maintain its immunologic and biologic activity.
The present invention provides antibodies directed
against the recombinant salivary allergens. These
antibodies can be used in immunoassays and for --
purification and standardization of allergens.
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Applicants are producing mAb directed against
recombinant allergens in BALB/c mice.
Antibodies (immunoglobulins) may be either
monoclonal or polyclonal and are raised against the
immunogen. Conveniently, the antibodies may be
prepared against the immunogen or part of the immunogen
for example a synthetic peptide based on the sequence,
or prepared recombinantly by cloning techniques or the
natural gene product and/or portions thereof may be
isolated and used as the immunogen. Such immunogens
can be used to produce antibodies by standard antibody
production technology well known to those skilled in
the art as described generally in Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1988 and
Borrebaeck, Antibody Engineering - A Practical Guide,
W.H. Freeman and Co., 1992. Antibody fragments may
also be prepared from the antibodies and include Fab,
F(ab')Z, and Fv by methods known to those skilled in the
art.
For producing polyclonal antibodies a host, such
as a rabbit or goat, is immunized with the immunogen,
generally with an adjuvant and, if necessary, coupled
to a carrier; antibodies to the protein are collected
from the sera. Further, the polyclonal antibody can be
absorbed such that it is monospecific. That is, the
sera can be absorbed against related immunogens so that
no cross-reactive antibodies remain in the sera
rendering it monospecific.
For producing monoclonal antibodies the technique
involves hyperimmunization of an appropriate donor with
the immunogen or immunogen fragment, generally from a
mouse, and isolation of splenic antibody producing
cells. These cells are fused to a cell having
immortality, such as a myeloma cell, to provide a fused
cell hybrid which has immortality and secretes the
required antibody. The cells are then cultured, in
bulk, and the monoclonal antibodies harvested from the
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culture media for use.
For producing recombinant antibody (see generally
Huston et al, 1991; Johnson and Bird, 1991; Mernaugh
and Mernaugh, 1995), messenger RNAs from antibody
producing B-lymphocytes of animals, or hybridoma are
reverse-transcribed to obtain complimentary DNAs
(cDNAs). Antibody cDNA, which can be full or partial
length, is amplified and cloned into a phage or a
plasmid. The cDNA can be a partial length of heavy and
light chain cDNA, separated or connected by a linker.
The antibody, or antibody fragment, is expressed using
a suitable expression system to obtain recombinant
antibody. Antibody cDNA can also be obtained by
screening pertinent expression libraries.
The antibody or antibody fragment can be bound to
a solid support substrate or conjugated with a
detectable moiety or be both bound and conjugated as is
well known in the art. (For a general discussion of
conjugation of fluorescent or enzymatic moieties see
Johnstone & Thorpe, Immunochemistry in Practice,
Blackwell Scientific Publications, Oxford, 1982.) The
binding of antibodies to a solid support substrate is
also well known in the art. (see for a general
discussion Harlow & Lane Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Publications, New
York, 1988 and Borrebaeck, Antibody Engineering - A
Practical Guide, W.H. Freeman and Co., 1992) The
detectable moieties contemplated with the present
invention can include, but are not limited to,
fluorescent, metallic, enzymatic and radioactive
markers such as biotin, gold, ferritin, alkaline
phosphatase, ~i-galactosidase, peroxidase, urease,
fluorescein, rhodamine, tritium, 1'C and iodination.
Additionally, in the production of the antibody, a
mimetope can be used as the antigenic source. That is
a molecule having an epitope the same or similar to tl~e
antigenic determinant (epitopes) of interest may be
CA 02309387 2000-OS-12
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used as the source of the eliciting antigen.
The diagnosis of Skeeter Syndrome should be
considered in any patient with cellulitis (a localized
area of erythema edema, induration, pain and/or itch)
at the site of a mosquito bite and negative cultures.
Appropriate antibiotic treatment should be continued
until a bacterial etiology for the inflammation has
been ruled out with certainty; then, topical and/or
oral glucocorticoids and an H,-antagonist can be
substituted.
While the long-term prognosis of Skeeter Syndrome
is generally favorable, natural desensitization depends
on the frequency and intensity of exposure and may take
many years to develop.
The antigens in the kit of the present invention
are found in the saliva of the mosquitoes Aedes vexans
and Aedes aegypti, and are cross-reactive with other
mosquito species. They have been characterized in our
laboratory as follows:
Antigens:
There are more than 12 antigens in the saliva of
Aedes vexans. Their molecular weight ranges between
17.5 and 75 kDa. There are more than eight antigens in
the saliva of Aedes aegypti. Their molecular weight
ranges between 18.5 and 68 kDa.
Antibodies:
Mosquito saliva antigens induce IgE, IgG, and
especially IgG4 antibody responses which mediate the
immediate and delayed type hypersensitivities in
humans. These antibodies are found in patients with
inflammatory reactions to mosquito bites (Skeeter
Syndrome).
Relevance to Human Allergic Disease:
The Aedes vexans or Aedes aegypti salivary
antigens are used in the ELISA immunoassays for
measurement of mosquito saliva-specific IgE, IgG, and __
IgG4 antibodies, as known in the art.
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These assays are applicable to patients presenting
with rashes and other symptoms after mosquito bites,
especially those with erythema, edema, and induration,
pain or itch at the sites) of mosquito bite(s), with
or without fever. Such patients are at risk for severe
localized inflammatory reactions and systemic reactions
to mosquito bites.
We expect such patients to have antibodies in the
range of 30 - 5,000 U/ml for specific IgE, 30 - 7,000
U/ml for specific IgG, and 50 - 33,000 U/ml for
specif is IgG4 .
The above discussion provides a factual basis for
the use of recombinant mosquito salivary proteins for
use in skin testing and immunotherapy. The methods
used with and the utility of the present invention can
be shown by the following non-limiting examples and
accompanying figures.
EXAMPLES
GENERAL METHODS:
Most of the general techniques described
hereinbelow are widely practiced in the art, and most
practitioners are familiar with the standard resource
materials which describe specific conditions and
procedures. However, for convenience, the following
paragraphs may serve as a guideline.
Geaeral methods in molecular biology: Standard
molecular biology techniques known in the art and not
specifically described were generally followed as in
Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Springs Harbor Laboratory, New York
(1989), in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore,
Maryland (1989) and in Perbal, A Practical Guide to
Molecular Cloning, John Wiley & Sons, New York (1988).__
General methods in immunology: Standard methods in
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immunology known in the art and not specifically
described were generally followed as in Stites et
al.(eds), Basic and Clinical Immunology (8th Edition),
Appleton & Lange, Norwalk, CT (1994) and Mishell and
Shiigi (eds), Selected Methods in Cellular Immunology,
W.H. Freeman and Co., New York (1980).
Laboratory-made mosquito Aedes aegypti preparations:
An Aedes aegypti mosquito colony was obtained from the
Department of Entomology, University of Manitoba, and
maintained in applicants' laboratory. Female
mosquitoes were used in the preparation of mosquito
whole body, head and thorax, salivary gland, and saliva
extracts. Saliva was collected from 3-15 day old adult
mosquitoes by placing the proboscis of each mosquito
into a capillary tube filled with water. Salivation
was induced by applying 0.5 ~l of 0.5% malathion in
acetone to the thorax [Boorman, 1987]. One hour later,
the contents of capillary tubes containing saliva were
collected, pooled, and lyophilized. The saliva was
reconstituted by dissolving the lyophilized proteins in
0.02 M phosphate buffered saline. In general, the
protein concentration was 0.6 mg/ml for Ae. vexans
extract, 0.4 mg/ml for Cx. quinquefaciatus extract, and
0.3 mg/ml for Ae. aegypti extract as measured by a Bio-
Rad Protein Assay kit (Bio-Rad Labs, Richmond, CA).
Skin bite tests and blood samples: Skin bite tests were
performed with one Ae. vexans mosquito and one Ae.
aegypti mosquito on the volar aspect of each subject's
forearm as previously described (Peng et al., 1996].
The wheal and flare circumferences were traced at 20
minutes and 24 hours after the bite, using a felt-
tipped pen. All wheal and flare tracings were
transferred to transparent paper. The area of the
wheal, flare or induration was measured using an IBM-PC
(IBM Instruments, Inc., Danbury, Conn.) digitizer and
stereometric measurement software [Sirnons, et al, --
1990] .
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A wheal of less than 0.3 cm2 with no flare and no
itch was considered to be a negative immediate
reaction. An induration less than 0.3 cm2 was considered
to be a negative delayed reaction.
Preparation of mosquito salivary gland extract:
Mosquitos (Aedes vexans), collected and identified in
the Department of Entomology, University of Manitoba,
were anaesthetized by chilling them at 4°C in a
refrigerator. Salivary glands were dissected from
female mosquitos in 0.02 M phosphate buffered saline
(PBS), pH 7.2 under a binocular microscope and
immediately transferred to 1 ml of PBS on ice. A total
of 370 salivary glands were gathered in l.ml of PBS,
ultrasonicated for 30 seconds, and centrifuged at 8820
g for 15 minutes. The supernatant was collected,
aliquoted, and stored at -70°C. The protein
concentration of the antigen preparation was 0.6 mg/ml
as determined by the Lowry method.
Measurement of human serum mosquito-specific IgE and
IgG by ELISA: Mosquito-specific IgE and IgG in human
sera were measured using indirect ELISAs.
Standardization of ELISA results between assays and the
estimation of relative amount of mosquito-specific IgE
or IgG in each sample was accomplished by using
reference IgE and IgG sera. These reference sera were
obtained from one subject with a high value of
mosquito-specific IgE and another subject with a high
value of mosquito-specific IgG and were defined as
1,000 U/ml for mosquito-specific IgE and IgG
antibodies, respectively. Polystyrene immunoplates
(Nunc-Immuno Plate Maxisorp, Denmark) were coated with
mosquito salivary gland extract (0.05 ~tg/well) diluted
in 0.05 M carbonate buffer (pH 9.6) and incubated
overnight at 4°C. After rinsing the plates three times
with washing buffer (0.05% Tween 20 in 0.01 M PBS, pH
7.2) and blocking them with 2% bovine serum albumen --
(BSA) in washing buffer for one hour at room
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temperature, 0.1 ml of the test sample or of the
reference serum diluted in ELISA buffer (0.2% BSA in
washing buffer) was added to each well. The plates
were incubated overnight at room temperature for IgE
and 1.5 hours at 37°C for IgG. The wells were washed,
and 0.1 ml of goat anti-human IgE (P. S. myeloma-
affinity purified, a gift from Dr. N. F. Adkinson, Jr.,
The Johns Hopkins Allergy and Asthma Centre, USA) or
goat anti-human IgG Fc fragment (Jackson ImmunoResearch
Laboratories, Inc., PA, USA) diluted in ELISA buffer
was added and incubated for 1.5 hours at 37°C. After
washing, 0.1 ml of diluted enzyme-conjugated rabbit
anti-goat IgG (Jackson ImmunoResearch Laboratories,
Inc.) was added and incubated for 1.5 hours at 37°C.
After a final wash, 0.1 ml of the enzyme substrate (1
mg/ml of p-nitrophenylphosphate in diethanolamine
buffer, pH 9.8) was added and incubated overnight at
4°C. The reaction was stopped by addition of 0.1 ml of
1 N NaOH. Optical absorbance at 410 nm was read, using
the THERMOmax microplate reader (Molecular Devices, CA,
USA). The value of mosquito-specific antibodies was
calculated by interpolation from the dilution curve of
the reference serum.
Both IgE and IgG determinations were performed on
two dilutions of serum, with each dilution duplicated.
50 samples were assayed twice for mosquito-specific IgE
and IgG.
Inhibition teats: In order to examine the specificity
of the assays, inhibition tests were conducted [Pang et
al, 1995]. The mosquito salivary gland extract was 10-
fold sequentially diluted in ELISA buffer. Each
dilution of the extract was incubated with a diluted
serum with high mosquito-specific IgE or high mosquito-
specific IgG for two hours at 37°C and then followed by
4°C overnight. The final serum dilution was one in 20
for the inhibition of IgE and one in 200 for that of --
IgG. Incubation of the serum dilution with ELISA
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buffer served as a control. Incubations of the diluted
serum with 10-fold serial dilutions of grass pollen
(Hollister-Stier, Miles Canada Inc., Ont.) also served
as controls. Mosquito-specific IgE and IgG in these
samples were then measured by ELISA as described above.
these tests validated the ELISA immunoassays [Peng et
al, 1995].
Statistical analysis: Analysis of data was performed
using NCSS software. Unpaired t tests were used for
between group comparisons. Linear regressions were
used for the correlation of serum mosquito-specific IgE
and IgG antibodies.
SDS-PAGE and silver stain: Proteins from mosquito
extracts were separated by SDS-PAGE under reducing
conditions in a discontinuous system using a Bio-Rad
mini slab,gel apparatus. One to two micrograms each of
the laboratory-made mosquito whole body, head and
thorax, salivary gland, and saliva extracts, were
loaded onto different wells and electrophoresed in 10%
acrylamide SDS-PAGE. Molecular weight protein
standards (Bio-Rad) were used to determine the relative
molecular weights of the electrophoresed components.
Separated proteins were detected by silver staining
(Bio-Rad Silver Stain kit).
SDS-PAGE and iacmnunoblot analysis for IgE and IgG
binding antigens: The proteins in the mosquito extracts
were separated by SDS-PAGE in a discontinuous system
according to Laemmli [1970] using a Bio-Rad slab gel
apparatus. For each mosquito extract, fifteen
micrograms of proteins prepared in a reducing buffer
were loaded onto each well and separated by
electrophoresis in 12% SDS-PAGE. These proteins were
then electro-transferred onto nitrocellulose membranes.
Free binding sites on the membranes were blocked by
incubation with 3% bovine serum albumin (Sigma, St.
Louis, MO) dissolved in 0.02 M PBS for two hours.
Immunoblot was completed by incubation of the
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membranes with a pooled serum exhibiting high mosquito-
specific IgE and IgG (1:10 dilution for IgE and 1:50
for IgG) over night. This was followed by incubation
with monoclonal anti-human IgE (1:15,000) (ascites,
clone No. 7.12, a gift from Dr. A. Saxon, Univ. of
California) or monoclonal anti-human IgG (1:15,000)
(PharMingen, CA) for 1.5 hours. After washing three
times with PBS containing 0.050 (v/v) Tween 20, the
membranes were incubated with horseradish peroxidase
l0 conjugated goat anti-mouse IgG (1:5,000 dilution for
IgE and 1:10,000 for IgG) (Calbiochem Corporation, CA)
for 1.5 hours. After washing, the membranes were
incubated in ECL detecting reagents (Amersham Life
Science, Buckinghamshire, England)) and then exposed to
Kodak film (X-Omat, Kodak).
PBS and umbilical cord serum were used
respectively to replace the pooled human serum as
controls. Pre-stained SDS-PAGE standards (Bio-Rad,
Richmond, CA) were used to determine the relative
molecular weights of the electrophoresed components.
EXAMPLE 1
COMPARISON OF PROTEINS, IgE AND IgG BINDING
ANTIGENS, AND SKIN REACTIVITY IN COMMERCIAL AND
LABORATORY-MADE MOSQUITO EXTRACTS
In order to improve the precision of diagnosis of
mosquito allergy, and to ensure the efficacy and safety
of imrnunotherapy with mosquito extracts, applicants
previously analyzed commercial mosquito extracts with
respect to protein and antigenic composition, and
biologic activity [Peng and Simons, 1995]. Seven
commercially available mosquito whole body extracts
supplied for skin testing and/or immunotherapy from six
manufacturers were investigated. The commercial
materials were compared with four mosquito extracts
made in applicants' laboratory from mosquito whole
body, head and thorax, salivary gland, and saliva. --
Epicutaneous tests, measurement of protein
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concentration, sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) for protein components,
and SDS-PAGE and immunoblotting for IgE and IgG binding
antigens were performed with all commercial and
laboratory-made extracts.
Commercial mosquito extracts: Seven commercial mosquito
extracts available for epicutaneous tests and/or
immunotherapy in mosquito allergy were purchased from
six manufacturers. These extracts were made from Cule
and Aedes genera. Information provided by the
manufacturers about the extracts is listed in Table 1.
Epicutaneous tests and mosquito bite tests:
Epicutaneous tests with the seven commercial mosquito
extracts and the four laboratory mosquito preparations
were performed on two subjects with severe skin
reactions to Aedes aegypti bites and on two subjects
with no skin reaction to the bites. Histamine
phosphate (1 mg/ml), saline and 50% glycerin in saline
were used as positive and negative controls,
respectively. Mosquito bite tests were performed using
female mosquitoes (Aedes aegypti) reared in the
laboratory. Skin immediate wheal and flare reactions
were measured 30 minutes after the tests and skin
delayed papule reactions were measured 24 hours later
using the largest and the orthogonal diameters of the
wheal or the papule, respectively. The area of the
wheal or papule was calculated after subtracting the
area of the wheal or papule produced by the relevant
negative control (if present).
Protein assay: Protein concentration of each commercial
mosquito extract and laboratory mosquito preparations
was determined by a Bio-Rad Protein Assay kit (Bio-Rad
Labs, Richmond, CA).
SDS-PAGE and immunoblot analysis of IgE and IgG bindiag
antigens: The pooled serum used in immunoblotting was
obtained from six subjects exhibiting severe skin --
reactions to mosquito bites and high mosquito-specific
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IgE and IgG as measured by ELISA. These subjects,
including the two skin and bite test positive subjects,
lived in Manitoba or Texas where the Aedes and/or Culex
mosquito species are abundant.
In the comparison of commercial mosquito extracts,
~,1 of each commercial extract, 6 ~,1 of Aedes aegypti
saliva and 10 ~.1 of Culex quinquefaciatus salivary
gland extract were loaded onto different wells.
Proteins separated by 12% SDS-PAGE were
10 electrophoretically transferred onto nitrocellulose
membranes. Free binding sites on the membranes were
blocked by incubation with 3% bovine serum albumin in
0.05 M PBS Tween 20 for two hours. Immunoblotting was
completed by incubation of the membranes with the
pooled serum (1:10 dilution for IgE binding antigens,
1:50 for IgG binding antigens). After washing, this
was followed by sequential incubations of the membranes
with monoclonal anti-human IgE or monoclonal anti-human
IgG (PharMingen, CA), and HRP-conjugated goat anti-
mouse IgG (Calbiochem Corporation, CA). After washing,
the membranes were finally incubated with ECL detecting
reagents (Amersham Life Science, Buckinghamshire,
England) and then exposed to the film (X-Omat, Kodak).
Pre-stained SDS-PAGE standards (Bio-Rad, Richmond, CA)
were used to determine the relative molecular weights
of the electrophoresed components.
RESULTS
Epicutaneous tests and protein concentrations:
Immediate and delayed reactions were found at the sites
of skin tests and mosquito bites in the two subjects
allergic to mosquito bites. The immediate reaction was
a pruritic wheal with a surrounding flare appearing
within a few minutes, peaking at 30 minutes and then
subsiding. The delayed papules were found several
hours later, reaching a peak 24 hours after the
epicutaneous and the bite tests. In contrast, there __
was little or no skin reaction to either the
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epicutaneous sites or the bite site in the two control
subjects.
Wide variations in both skin immediate and delayed
reactions to the seven commercial mosquito materials
were found in the two reactive subjects. The mean
immediate wheal sizes ranged from 0.5 to 32 mm~, and the
mean delayed papule sizes ranged from 0 to 36 mm~ (Table
2). Two aqueous extracts, C5 and C7, appeared to have
no biological activity in humans. The protein
concentration of the commercial extracts varied
greatly, ranging between 0.09 and 4.85 mg/ml,.but
generally correlated with the size of skin reactions (r
- 0.72, p < 0.003 fox wheal; r = 0.80, p < 0.005 for
papule). An exception to this was the C7 extract which
had a protein concentration of 1.67 mg/ml, but elicited
no skin response.
In order to evaluate the biological activity of
each gram of protein, the ratio of skin
reactions/protein concentration was calculated (Table
2). The skin reactivity per gram of protein varied
from 0.3 to 14.9 in the seven commercial extracts.
Among the four laboratory-made mosquito preparations,
as the purity of the preparation increased, rank
ordered from whole body, head and thorax, salivary
gland to saliva, the skin reactivity per gram of
protein increased significantly. Perhaps the skin
reactivity per gram of protein in saliva (78.0) was
less than that found in salivary gland (128.6) because
the lyophilization required in the preparation of
saliva extract reduced some biological activity of
saliva.
SDS-PAaE and silver stain: In the four laboratory
preparations, rank ordered from whole body, head and
thorax, salivary gland to saliva extracts, the amount
35~ of salivary antigens significantly increased, while
non-salivary proteins and antigens significantly
decreased. There were 24 visible protein bands in the
head and thorax extract, 16 in the salivary gland
CA 02309387 2000-OS-12
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extract, but only nine in the saliva extract. When
commercial extracts containing the same amount of
protein were used, the protein bands were obscure and
the background was very dark.
SDS-PAGE and immunoblot analysis: The IgE and IgG
binding antigens in the seven commercial mosquito
extracts were measured. The antigens of the three
commercial Aedes extracts were compared to the antigens
in the Aedes aegypti saliva extract, while the antigens
of the four commercial Culex extracts were compared to
the antigens in the Culex quinquefaciatus salivary
gland extract. Culex quinquefasciatus and Culex
pipiens belong to the same genus and are sibling
species. Because the Culex species does not salivate
using the method applied to induce Aedes species to
salivate, the salivary gland extract of Culex
quinquefaciatus was used to compare antigens with the
Culex pipiens whole body extracts. Among the seven
commercial mosquito extracts both IgE and IgG binding
antigens varied greatly both in the number of antigen
bands and the amount of each antigen. Multiple
antigens were found in the commercial extracts. Most
of these antigens were not present in the saliva or
salivary gland extract. Some extracts, C1, C2, C3,
contained small amounts of saliva antigens. Two
extracts, C5 and C7, contained no visible saliva or
salivary gland antigens, although they did contain
other proteins (0.09 and 1.67 mg/ml of proteins,
respectively).
The IgE and IgG binding antigens in one commercial
mosquito whole body extract and four laboratory-made
mosquito preparations (whole body, head and thorax,
salivary gland and saliva) were observed. Whole body,
head, and thorax extracts contained multiple antigens __
which were not present in saliva, but they contained
few of the antigens present in saliva, although more
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micrograms of proteins were loaded for these extracts.
As the purity of the preparation increased from whole
body extract to saliva, the number of proteins and
antigens decreased dramatically. This observation
correlates with the results shown in Table 2, in which
the salivary gland and saliva extracts have lower
protein concentrations (0.14 and 0.25 mg/ml,
respectively) and higher ratios of skin
reactivity/protein (128.6 and 78.0, respectively) than
those in the whole body (protein 1.2 mg/ml, ratio 9.2)
and the head and thorax extracts (protein 0.62 mg/ml,
ratio 17.7).
DISCUSSTON: Using both in vitro and in vivo tests,
considerable variation was found in the commercial
mosquito extracts. Indeed, two (C5 and C7) of three
aqueous extracts (C4,C5,C7) elicited neither immediate
nor delayed skin reactions and presented no visible
antigen band in immunoblot. All four extracts
preserved by 50% glycerin exhibited skin reactivity and
visible protein bands suggesting that the extracts
preserved in 50°s glycerin are more potent and stable.
Commercial mosquito extracts contained multiple
proteins and antigens, many of which are unrelated to
the antigens in mosquito saliva. The antibodies
observed in the human subjects directed against these
non-saliva antigens in the commercial extracts may have
been induced by inhalation of insect particles or by
being bitten by other insects whose antigens crose-
reacted with mosquito body components leading to the
formation of IgE and IgG antibodies against mosquito
body antigens.
These results suggested that commercial mosquito
extracts should be standardized and that purer mosquito
extracts should be used in the diagnosis and
immunotherapy of mosquito allergy. In.addition to _-
having poor diagnostic accuracy in skin testing,
injection of crude extract may lead to the development
of sensitization to some of its components (Hamilton,
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CA 02309387 2000-OS-12
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1990] .
Standardization of the extracts is required
because the process of collecting or extracting
mosquito saliva is extremely tedious, it is currently
impractical to uae mosquito saliva or salivary gland
extracts in diagnosis or immunotherapy. Therefore if
commercially available mosquito extracts are to be used
they must be standardized to increase the quantity of
the active materials they contain. Standardization of
antigen preparation is usually achieved by comparison
of overall activity or major components of the extract
with those of a reference preparation. An in-house
reference mosquito extract and a pooled serum for each
mosquito genus are used to evaluate the relative
biological activity and the lot-to-lot variation of
different batches of mosquito extracts including
species specific and non-specific components. However,
this procedure is not efficient and better means of
standardized extracts are required. The present
invention provides the use of recombinant salivary
allergens to be used as a "recombinant extract" to
simplify the need for standardization and to provide
greater safety in immunotherapy.
EXAMPLE 2
CROSS-REACTIVITY OF ERIN AND SERUM SPECIFIC IgE
RESPONSES AND ALLERGEN ANALYSIS FOR THREE MOSQUITO
SPECIES PITH WORLD-HIDE DISTRIBUTION
In order to improve diagnosis and immunotherapy of
mosquito allergy, purified or recombinant mosquito
saliva antigens should be used as shown in Example 1.
In order to do so, it should be determined if there are
any cross-reactive skin and IgE responses and species-
shared antigens among various mosquito species,
especially those with world-wide distribution.
Ae. vexans, Ae. aegypti, and Cx. quinquefasciatus
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are the three most important mosquito species
distributed globally. Ae. aegypti (see generally
Christophers, 1960. Aedes aegypti (L.) the yellow fever
mosquito: its life history, Bionomics and Structure.
London: Cambridge University Press) and Cx.
quinquefasciatus (see generally Knight and Stone, 1977.
A Catalog of the Mosquitoes of the World. 2nd ed.
Washington: Ent Soc Am (Thomas Say Found.)) are found
throughout the tropical regions of the world within
20°C isotherms, and Ae. vexans is found in North
America, Eurasia, Asia, and Africa [Wood et al, 1979].
Comparison of the human immunological response to the
three species has never been made. Because of climate,
Ae. aegypti and Cx. quinquefasciatus are not present in
Canada, while Ae. vexans is the major pest in Canada
representing up to 80% of the local mosquito
population. In order to determine whether the three
mosquito species have cross-reactive immunological
responses and species-shared antigens, skin bite tests
were performed and serum mosquito specific IgE was
evaluated for the three mosquito species in 41
Manitobans who had been exposed to Ae. vexans bites,
but not to Ae. aegypti and Cx. quinquefasciatus bites.
Species-shared allergens were also analyzed by
immunoblotting, using the sera from mosquito-allergic
subjects and an antibody to a recombinant Ae. aegypti
salivary protein.
MATERIALS AND METHODS
Subjects: This project was approved by The
University of Manitoba Faculty Committee on the Use of
Human Subjects in Research, and the participants gave
written, informed consent before study entry. Forty-
one healthy subjects (21 males and 20 females), age 19
to 57 years, with skin reactions to mosquito bites
ranging from none to strongly positive were recruited
during the summer of 1993. All the subjects had lived
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in Canada more than two years, and 71% had lived in
Canada (mostly in Manitoba) since birth.
Antihistamines and other medications which might
suppress the skin bite test were withheld for an
appropriate length of time before the study.
Mosquitoes mosquito saliva aad salivary gland
extracts: Female Ae. vexans mosquitoes were collected
in local fields and identified by scientists in the
Department of Entomology, University of Manitoba. The
Ae. aegypti colony was obtained from the same
Department and maintained in our laboratory. The Cx.
quinquefaciatus colony was imported from Dr. Robert J.
Novak's laboratory, University of Illinois, Champaign,
IL, and maintained in our laboratory. Four to twelve
day old adult Ae. aegypti mosquitos were used for the
bite tests and for saliva collection. Salivary glands
were dissected from four to twelve day old adult Cx.
quinquefaciatus mosquitoes. Mosquito saliva or
salivary gland extracts were prepared for use in the
ELISA and immunoblot as described herein above.
ELISA: Serum mosquito-specific IgE to Ae. vexans, Ae.
aegypti, and Cx. quinquefaciatus were measured by an
indirect ELISA as described herein above. Optimal
conditions for dilutions of the 3 mosquito extracts,
serum samples, goat anti-human IgE, and conjugated
rabbit anti-goat IgG were chosen by checkerboard
titration. Standardization of ELISA results between
assays and estimation of the relative amount of
mosquito-specific IgE in each sample was accomplished
by using reference sera as described. The reference
serum used to measure Ae. aegypti-IgE and Cx.
quinquefaciatus-IgE was obtained from a subject with
systemic reactions to mosquito bites (kindly provided
by Dr. R.J. Engler, Walter Reed Army Medical Centre,
Washington, D.C.). Another reference serum used to
measure Ae. vexans-IgE came from a Manitoban with --
CA 02309387 2000-OS-12
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severe skin reactions to mosquito bites (immediate
wheal 1.5 cm2 and flare 11.6 cmz). Both reference sera
were defined as 1000 U/ml for mosquito-specific IgE.
Microplates coated with mosquito saliva or salivary
gland extract (0.02 - 0.05 ~1g/well) were sequentially
incubated with serum samples (1:20) or reference serum
(2-fold dilutions from 1:20 to 1:10,240), 1:1,000 goat
anti-human IgE (P. S. myeloma-affinity purified, a gift
from Dr. N.F. Adkinson, Jr., The Johns Hopkins Allergy
and Asthma Centre), 1:1,000 alkaline phosphatase-
conjugated rabbit anti-goat IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA). The values of mosquito-
specific IgE in the tested samples were calculated by
interpolation from the dilution curve of the reference
serum. The sensitivity of the ELISAs was 0.8 U/ml.
ELISA inhibition tests: In order to study the cross-
reactivity among the three species, ELISA inhibition
tests were performed. A serum with high mosquito-
specific IgE (final dilution 1:20} was incubated with
serially diluted Ae. vexans, Ae aegypti, Cx.
quinquefaciatus extracts with final dilutions of 1:4
and 1:20 at room temperature for one hour and then 4°C
overnight. Incubation of the serum with ELISA buffer
served as a positive control. These incubated materials
were then measured for Ae. aegypti-IgE using ELISA.
Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and iamnunoblot analysis:
Among the 41 subjects studied, sera from six subjects
with large immediate skin wheal responses (> 1.0 cm2) in
the bite tests and high Ae. vexans-specific IgE levels
(> 1,000 U/ml) were pooled and used for immunoblotting.
SDS-PAGE and immunoblotting were performed as
described herein above and in Peng et al., 1996. In
the analysis of IgE and IgG antigens, two ~tg of the
proteins from each mosquito extract were loaded and
electrophoresed in 12% acrylamide SDS-PAGE under __
reducing conditions. Proteins separated by SDS-PAGE
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CA 02309387 2000-OS-12
_ WO 99!25826 PCT/IB98/01961
were electrophoretically transferred onto
nitrocellulose membranes. Free binding sites on the
membranes were blocked by incubation for two hours with
3% bovine serum albumin in 0.02 M phosphate buffered
saline, pH 7.4, containing 0.05% (v/v) Tween 20 (PBS-
T). After washing three times with PBS-T, the
membranes were incubated overnight with the pooled
serum (1:10 dilution for IgE and 1:50 for IgG) and
washed again. Incubation of the membranes with PBS-T
served as a negative control. This was followed by
sequential incubations with 1:15,000 monoclonal anti-
human IgE (clone 7.12, from Dr. A. Saxon's laboratory,
University of California) or 1:15,000 monoclonal anti-
human IgG (PharMingen, San Diego, CA), and then HRP-
conjugated goat anti-mouse IgG (1:5,000 for IgE,
1:10,000 for IgG) (Calbiochem Corporation, La Jolla,
CA). After washing, the membranes were finally
incubated with ECL detecting reagents (Amersham Life
Science, Buckinghamshire, England) and then exposed to
the film (X-Omat, Kodak). Pre-stained SDS-PAGE
standards (Bio-Rad, Richmond, CA) were used to
determine the relative molecular weights of the
electrophoresed components.
The same method, with some modifications, was used
in the study of a recombinant Ae. aegypti saliva
protein, which was kindly provided by Dr. A. James at
the University of California, Irvine, CA (James et al,
1991]. One ~tl of the baculovirus medium containing the
recombinant protein and 2 ~,g of each mosquito extract
were loaded onto different wells and electrophoresed.
Medium from cells infected with wild-type baculovirus
was used as a control. The membranes containing
separated proteins were then incubated with rabbit
antibody to the recombinant protein (1:5,000) (from Dr.
A. James) followed by incubation with HRP-conjugated
goat anti-rabbit IgG (1:5,000). The remaining steps
were the same as the immunoblot using human serum. '-
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Statistical analysis: Analysis of the data was
performed using the "Number Crunch Statistical System"
software. Unpaired t tests were used between group
comparisons. Linear regressions were used for the
analysis of correlations among skin reactions and serum
mosquito-specific IgE levels.
RESULTS
Skin bite reactions: No systemic reactions were noted
after the bite tests. Immediate and delayed skin
reactions to mosquito bites were observed. The
immediate reaction consisted of a pruritic wheal with a
surrounding flare or erythema appearing within several
minutes, reaching a peak at 20 - 30 minutes and then
subsiding. The delayed reaction consisted of an
indurated papule which appeared several hours later,
and was peaked 24 - 36 hours and diminished over
several days after the bite. In five subjects with
severe delayed reactions, vesicles were found in the
centres of the indurated areas, precisely in the area
where the immediate wheal had been.
Twenty nine of the 41 subjects had positive
immediate skin reactions to Ae. vexans bites. Twenty
two of the 29 also reacted to Ae. aegypti bites. Seven
subjects reacted to Ae. vexans bites only. The size of
the immediate reactions ranged from 0 to 3.1 cm2 for the
wheal and 0 to 23.0 cm2 for the flare, and 0 to 29.5 cm2
for delayed reactions.
In the immediate reaction, wheals correlated
significantly with flares in both species (r = 0.72, p
< 0.00 for Ae. vexans; r = 0.88, p < 0.00 for Ae.
aegypti). Also, immediate wheal and flare reactions
showed significant correlation with the delayed
reactions in each species as well (r's between 0.43 and
0.61, p's < 0.00). More interestingly, significant
correlations of skin reactions were found between the
two species, especially between the immediate wheal --
33
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sizes of the two species (r = 0.84, p < 0.00) and the
delayed induration reactions of the two species (r =
0.77, p < 0.00).
Mosquito-specific IgE levels: The geometric mean Ae.
vexans-IgE, Ae. aegypti-IgE, and Cx. quinquefaciatus-
IgE were all significantly higher in the subjects with
immediate skin reactions to Ae. vexans bites than in
those with no immediate skin reaction to the bites (p's
< 0.05). Similar results were found for mean mosquito-
specific IgE levels of the three species in the
subjects with or without immediate skin reactions to
Ae. aegypti bites (Figure 2 bottom). Significant
correlations were also found among the IgE levels of
the three species (r~s between 0.35 to 0.60, p~s <
0.03) .
Correlations among skin reactions and the IgE levels:
There was inter-correlations among skin reactions and
IgE levels of the three species. As expected, Ae.
vexans-IgE values correlated significantly with skin
reactions to Ae. vexans bites, and Ae. aegypti-IgE
values correlated significantly with skin reactions to
Ae. aegypti bites. Ae. vexans-IgE also significantly
correlated with skin reactions to Ae. aegypti bites.
The same correlation was found between Ae. aegypti-IgE
and the delayed skin reactions to Ae. vexans, and
between Cx. quinquefaciatus-IgE and both immediate and
delayed reactions to Ae. vexans bites.
ELISA inhibition tests: The cross-reactive
immunological responses among the three species were
further confirmed by ELISA inhibition tests. Ae.
aegypti-specific IgE reactions could be inhibited by
incubation of the serum with all three extracts in a
dose-dependent manner, confirming the existence of
species-shared antigens among the extracts of the three
species.
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SDS-PAGE and iamnunoblot analysis: Immunoblot analysis
further revealed the existence of species-shared
antigens. Using the pooled serum from Manitobans
allergic to mosquitos, the IgE and IgG antibodies not
only bound to Ae. vexans antigens, but also to the
antigens of the two species which are not found in
Manitoba. There were no antigen bands found in the PBS
control strips. Further, two bands in Ae. aegypti and
Cx. quinquefaciatus extracts and one band in Ae. vexans
extract were recognized by the rabbit antibody to the
recombinant protein, suggesting that the 37 Kda protein
of Ae. aegypti is also present in the salivary glands
of Cx. quinquefaciatus and Ae. vexans.
Cross-reactive IgE responses have not been
I5 previously reported in mosquito allergy. Unlike other
studies of cross-reactivity to insect bites or stings,
we selected a specific location where Ae. aegypti and
Cx. quinquefasciatus are not present, while Ae. vexans
is the major pest representing up to 80% of the
indigenous mosquito population. This allowed us to
exclude the sensitization caused by the other two
mosquito species.
The immunologic basis for the reactive skin and
IgE responses among different mosquito species is the
existence of species-shared antigens which are based on
their identical protein, sequences. Salivary secretions
have been demonstrated to be directly responsible for
skin reactions to mosquito bites. In the present
Example, immunoblot analysis using saliva or salivary
gland extracts, a number of species-shared antigens and
several Ae. vexans-specific antigens were found.
Using the antibody to the recombinant protein, it
was determined that the 37 kDa Ae. aegypti saliva
protein is present in Ae. vexans and Cx.
quinquefaciatus salivary gland extracts, further
confirming the existence of species-shared antigens in --
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the three species. These species-shared antigens may
well explain the cross-reactivity of skin reactions and
IgE responses among different mosquito species.
EXAMPLE 3
IMMLTNOBLOT ANALYSIS OF IgE AND IgG BINDING ANTIGENS IN
EXTRACTS OF MOSQUITOES AEDES VEXANS, CLTLEX TARSALIS AND
CULISETA INORNATA
Reactions to mosquito bites are generally caused
by immunologic mechanisms, with both type I (IgE-
mediated) and type IV (cell-mediated)
hypersensitivities being involved [Oka K, 1989; Peng et
al, 1996; Reunala et al, 1994a; 1994b]. Serum mosquito-
specific IgE has been demonstrated to correlate with
cutaneous mosquito bite reactions [Oka K, 1989; Peng et
al, 1996]. Mosquito-specific IgG has also been found to
correlate with skin mosquito bite reactions, suggesting
that IgG may also be involved in the development of
mosquito allergy [Peng et al, 1996].
Mosquito antigens have been identified by
immunoblot analysis. A number of mosquito antigens with
molecular masses ranging from 14 to 126 kDa have been
reported in various mosquito species [Penneys et al,
1989; Shen et al., 1989; Wu and Lan, 1989; Brummer-
Korvenkontio, 1990, 1994]. In this Example antigens are
analyzed using immunoblot techniques on three mosquito
species not previously examined; Aedes (Ae.) vexans, a
globally distributed species (and the major pest
species in Manitoba), and two North American species
Culex (Cx.) tarsalis and Culiseta (Cs.) inornata.
Subjects: This study was approved by The University of
Manitoba Faculty Committee on the Use of Human Subjects
in Research, and the subjects gave written, informed
consent before participation. Forty-two subjects with
a history of local reactions to mosquito bites were
recruited during the summer of 1993. Skin mosquito Ae. '-
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vexans bite tests were performed and serum mosquito
(Ae. vexans) salivary gland-specific IgE and IgG
antibodies (mosquito-specific IgE and IgG) were
measured by ELISA in 42 subjects. Three subjects with
severe skin reactions in the bite tests who also
exhibited high mosquito-specific IgE and IgG levels,
and two subjects with no skin reaction to the bites and
with low mosquito-specific antibody levels, were
selected for this study.
Skin reactions in the bite tests and antibody
levels in the five subjects studied are listed in Table
3. The reactive subjects had very strong skin immediate
reactions which consisted of a pruritic wheal (z 1 cm)
with surrounding flare appearing within several
minutes, reaching a peak at 30 minutes and then
subsiding. The immediate reaction was followed by a
local pruritic and indurated papule and erythema (z 7
cm) which appeared several hours later, reaching a peak
24 hours after the bite. The papule usually lasted
several days.
A pooled serum from reactive subjects was used in
the analysis of antigens in the three mosquito species.
Mosquito head and thorax extracts: Extracts prepared
from mosquito heads and thoraxes were used in the
study. Female mosquitoes of the three species (Ae.
vexans, Cx. tarsalis, Cs. inornata) were collected and
identified by the Insect Control Branch, Parks and
Recreation Department, City of Winnipeg, and then
stored at -70°C. After the abdomens, wings and legs
were removed, the heads and thoraxes were placed in
cold 0.02 M phosphate buffered saline (PBS), pH 7.2,
homogenized on ice for 1 minute using a PCU 11
homogenizes (Kinematica, Switzerland), centrifuged at
8820 g for 30 minutes, filtered through a 0.45 ~m
Amicon filter, and then stored at -70°C. The protein
concentrations of the antigen preparations were 1.48
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mg/ml for Ae. vexans, 6.13 mg/ml for Cx. tarsalis and
5.34 mg/ml for Cs. inornata, as measured by the Protein
Assay Dye Reagent kit (Bio-Rad Laboratories, Richmond,
CA) .
SDS-PAGE and immunoblot analysis for IgE and IgG
binding antigens: As described herein above.
RESULTS
Inhibition test: To examine the specificity of the
immunoblot analysis, an antigen inhibition test was
performed. Prior to immunoblotting, the pooled serum
with high mosquito-specific IgE and IgG was incubated
with Ae. vexans head and thorax extract with a final
dilution of 1:2, 1:20 or 1:200 at 4°C overnight.
Incubation of the serum with PBS served as a positive
control. SDS-PAGE and immunoblotting for IgE and IgG
binding antigens was then performed with these pre-
incubated serum samples. After incubation with
mosquito extract, both IgE and IgG binding bands were
significantly reduced compared to the positive control.
This inhibition exhibited dose-dependency demonstrates
that the immunoblot analysis of IgE and IgG binding
antigens is specific to mosquito antigens.
Antigens in the three mosquito species: IgE and IgG
antibodies bound to various mosquito antigens in the
three species. Twelve antigens in Ae. vexans, 16
antigens in Cx. tarsalis, and 14 antigens in Cs.
inornata, with molecular masses ranging from 18.5 to
160 kDa, were found by immunoblot analysis (Table 4).
Most of the antigens bound to both IgE and IgG, and
were shared by species. Nine antigens (24, 32.5, 40,
46, 50, 62, 65, 110, 160 kDa) were shared by three
species, especially the 40 kDa antigen. Six antigens
(28, 37, 43, 56, 70, 85 kDa) were shared by two
species. Only three antigens were species-unique: the
18.5 kDa in Ae. vexans, the 25 kDa in Cs. tarsalis, and
the 30 kDa in Cs. inornata. --
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igr: ann 1gG antibody responses to Ae. vexans antigeas
in 5 subjects with or without skin reactions to
mosquito bites: Figure 3 shows the IgE (left) and IgG
(right) responses to the antigens of Ae. vexans in
three subjects with severe skin reactions to the bites
(strip #1-3) and two subjects without skin reactions to
the bites (strips #4 and #5). In the five subjects, the
patterns (spectra) of IgE and IgG responses to the
antigens were similar, but the magnitudes of the
antibody responses varied suggestively. Significantly
strong IgE and IgG responses to the antigens,
especially to the 32.5, 40, 43, and 50 kDa antigens,
were found in the 3 reactive subjects (#1-3), while
only very faint IgE antibody responses were observed in
the 2 non-reactive subjects (#4,5), and very faint IgG
responses to the antigens in 1 non-reactive subject
(#5) .
The patterns of IgE and IgG responses to the
antigens varied slightly among the three reactive
subjects. Some individuals reacted to certain antigens
while others reacted to different antigens, eg. only
subject #3's IgE bound to the 43 kDa antigen, and the
IgE of subject #2 was the only one to bind to the 80
kDa antigens (Figure 3, indicated by arrows).
No antibody binding was found when the antigen
containing membranes were incubated with either the
cord serum (#6) or PBS (#7).
Comparison of IgE and IgQ responses in three subjects:
In order to define any differences between IgE and IgG
responses to mosquito antigens in the same individual,
the immunoblot results displaying IgE and IgG binding
antigens were placed side by side for the three
subjects with high mosquito-specific IgE and IgG
antibodies (Figure 4). Although the patterns of
antibody responses were slightly different among the
three subjects, all the antigens induced both IgE and
IgG responses. --
In summary, extracts of one globally distributed
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mosquito species (Aedes vexans) and two North American
species (Culex tarsalis and Culiseta inornata) were
prepared from heads and thoraxes. Proteins of the three
extracts were separated by 12% SDS-PAGE and transferred
to nitrocellulose membranes for immunoblotting.
Immunoblotting was completed by sequential incubations
of the membranes with a pooled human serum from su-
bjects allergic to mosquito bites, monoclonal anti-
bodies to human IgE or IgG, and goat anti-mouse IgG
conjugate. Twelve to sixteen antigens with molecular
masses ranging from 18.5 to 160 kDa were found in each
extract. Nine antigens were shared by three species and
six were shared by two species. Only three were
species-unique. Most antigens bound to both IgE and IgG
antibodies.
IgE and IgG antibodies against Aedes vexans were
studied by immunoblotting using individual serum from
subjects with or without skin reactions to Aedes vexans
bites. All three subjects with severe skin reactions
had strong IgE and IgG antibodies to 32.5, 40, and 50
kDa proteins. The patterns and magnitudes of IgE and
IgG antibodies to the antigens varied among
individuals. Very faint IgE antibodies to these
antigens were found in the 2 subjects with no skin
reactions, suggesting that IgE plays a role in the
development of mosquito allergy.
EXAMPLE 4
ISOLATION OF A CDNA ENCODING A 30 kDa IgE-BINDING
PROTEIN OF MOSQUITO AIDES AEGYPTI SALIVA
Isolation of cDNA close: Using mouse antiserum against
Aedes aegypti saliva, produced as previously described
[Yang et al., 1997], with alkaline phosphatase-
conjugated goat anti-mouse IgG (Jackson ImmunoResearch
Lab. Inc, West Grave, PA, USA) a salivary gland cDNA --
library of mosquito Aedes aegypti (kindly provided by
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Dr. A. James, University of California, Irvine) was
screened according to the lambda library protocol
(Clontech lab, CA, USA). Thirty-nine clones were
isolated from about 120,000 plaques. These clones were
grouped into A, B and C, according to the color
strength of their reactions to the mouse antiserum on
the filters. Four clones from each group were chosen
to prepare fusion proteins using the method previously
described [Huynh et al., 1985].
Twelve samples of the fusion protein were
separated by SDS-PAGE and immunoblotted by mouse anti-
saliva serum. A protein band with a molecular weight
ranging from 125 to 175 kDa was found in each sample
(data not shown). As the 13-galactosidase part of the
fusion protein is 114 kDa, the cDNA coding part ranged
from 10 to 60 kDa. Three clones with different size of
cDNA inserts were subcloned into pBluescript II SK
vector (Stratagene, La Jolla, CA, USA) and sequenced by
the Sanger method [1977] using a US Biochemicals
sequencing kit (Cleveland, Ohio, USA). One clone
(AA22) with a 0.75 kb of insert has a complete 3'
terminus, which is consistent with known eukaryotic
genes, a consensus polyadenylation sequence, AATAAA,
and polyadenosines (Table 4; SEQ ID Nos:l). An open
reading frame for 217 amino acids (estimated to be 25
kDa) was observed in frame with the B-galactosidase
protein, but this clone lacked an initiation codon in
the 5' terminal sequence.
PCR was designed to clone the 5' terminal fragment
from the cDNA library using the lambda gtll forward
primer (5' GACTCCTGGAGCCCG 3', Clontech; SEQ ID No:2)
and a synthesized 3' primer (3'ATATCTGTCCACCAACG 5';
SEQ ID No:3) complementary to a sequence of the 3'
terminal fragment. PCR was performed in 100 ~1 of the
sample containing 0.5 ~.g of library DNA, 10 ~1 of 10x
buffer, 2 ~l of 25 mM dNTP's, 2 ~1 of 100 ng/~1 each __
primer, 2 U of Taq polymerase supplied by the PCR kit
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(Boehringer Mannheim Canada, Quebec). The reaction was
subjected to 25 cycles of amplification consisting of 1
minute at 94°C, one minute at 55°C and one minute at
72°C, with a final 72°C extension for seven minutes.
The PCR product was cloned into the TA vector
(Invitrogen, San Diego, CA, USA).
Three PCR clones were obtained and sequenced. The
sequences of the three clones were found to be
identical and overlapped AA22 cDNA. In the search for
an initiation codon in the same apen reading frame as
AA22 cDNA, an ATG was found, which was characterized
with an adenosine at the crucial -3 position of the
Kozak consensus sequence, A/GXXXATG, for initiation of
translation by eukaryotic ribosomes (Kozak, 1987]. The
sequence flanking the putative translational start
sites, GAAAATG, is very similar to the consensus
sequence, C/AAAA/CATG, for initiation of Drosophila, an
insect gene (Gavener, 1987]. The full-length cDNA is
0.85 kb, coding for a protein of 253 amino acid
residues, approximately 30 kDa.
Characterization of the eDNA coding protein: Specific
mouse and human antibodies to the cDNA coding protein
were prepared using the methods previously reported
[Lyon et al., 1986; Caraballo et al., 1996].
Nitrocellulose filters were saturated with the fusion
protein by overlaying the filters onto the lawn of E.
coli Y1090 which had been previously infected by lambda
AA22, and then incubated with mouse anti-Aedes aegypti
saliva serum or a pooled mosquito-allergic human serum.
Fusion protein-selected mouse or human antibodies were
eluted from the filters by incubation of the filters
with glycine buffer. These antibodies were used to
probe the saliva proteins of Aedes aegypti. As shown in
Figure 1, many bands were revealed in the
immunoblotting with mouse anti-saliva serum (lane 1).
When immunoblotting was completed with the fusion --
protein-selected mouse antibodies, only a 30 kDa
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protein was specifically revealed (Figure 1, lane 2).
The fusion protein-selected mouse antibodies recognize
the 30 kDa saliva protein, indicating that this cDNA
clone encodes the 30 kDa saliva protein.
As shown in the Examples, the 30 kDa saliva
protein of mosquito Aedes aegypti elicited an IgE
response in 42% of mosquito-allergic subjects and in
none of the subjects without skin reactions to the
bites. In Figure 2, the fusion protein-selected human
IgE strongly bound to the 30 kDa native protein (lane
2), suggesting that the cDNA isolated codes the 30 kDa
salivary allergen which induces a specific IgE response
in mosquito-allergic humans. This IgE-binding protein
is the third salivary allergen of Aedes aegypti whose
cDNA has been cloned and sequenced.
From the searches of the DNA and protein databases
to determine the identity of the cloned cDNA using
BLAST network service [Altschul et al., 1990], it is
apparent that this putative protein represents a novel
protein. Although the BLASTN results based on the
nucleotide sequence indicate a high degree of
similarity to a number of known sequences, these
similarities most likely result from a number of
repetitive codons in the sequence (data not shown).
This interpretation is confirmed by the BLASTP search
based on the conceptual translation product of the
cDNA, which does not indicate any similarity to a known
protein. The protein is rich in glutamic acid residues
(16.5% of amino acid residues), and has a hydrophobic
amino terminal region characteristic of a secretory
signal peptide [Hopp et al., 1981; Kyte et al., 1982].
EXAMPLE 5
EXPRESSION, PURIFICATION, IMMUNOLOGICAL
CHARACTERIZATION, AND CLINICAL USE OF rAed a 1,
A 68 kDa RECOMBINANT SALIVARY ALLERGEN OF MOSQUITO
AEDES AEGYPTI
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Mosquito salivary proteins cause allergic
reactions in humans. Aed a 1, a 68 kDa mosquito Aedes
aegypti salivary protein, is an allergen which binds to
the IgE of mosquito-allergic subjects. In this
Example, an expressed, purified recombinant Aed a 1
(rAed a 1) was characterized to determine if it bound
to antibodies directed to the native protein.
Additionally, responses to it in mosquito-allergic
subjects was investigated to determine its biologic
activity, that is activity in vivo to elicit an immune
response.
Two cDNA segments were ligated together forming
the full-length Aed a 1 gene, which was inserted into
the baculovirus expression vector pBlueBacHis C.
Recombinant baculoviruses were generated by co-
infection of Sf9 insect cells with wild-type
baculovirus AcMNPV DNA and the recombinant vector. By
Western blot using rabbit anti-rAed a 1, the resultant
baculovirus were proved to express the 68 kDa rAed a 1
which was secreted into the culture medium as a non-
fusion protein. Also, by Western blot the recombinant
Aed a 1 showed identical immunological reaction with
the native Aed a 1 in the saliva. rAed a 1 in the
culture medium was then purified using anion exchange
and gel filtration chromatography.
Skin epicutaneous tests with purified rAed a 1 and
a commercial crude Ae. aegypti extract were performed
in 31 subjects with positive reactions in Ae. aegypti
bite tests and 17 subjects with negative reactions in
the bite tests (Figure 5). Immediate wheal and flare
were measured 20 minutes after the epicutaneous test,
and delayed reactions were measured 24 hours later.
Nine of 31 mosquito allergic-subjects (29%} had a
positive immediate reaction to rAed a 1, compared to 10
for the commercial extract (31%). In the nine subjects
with positive rAed a 1 reactions, the flare sizes
induced by rAed a 1 significantly correlated with those
induced by mosquito bites (r = 0.88, p < 0.001) (Figure
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6). Also, six of the nine subjects (18%) developed an
positive delayed skin reaction to rAed a 1, versus four
for the commercial extract (12%). None of the subjects
with negative reactions to mosquito bites (0%) showed a
positive immediate or delayed reaction to either of the
two mosquito preparations.
This Example demonstrates that the recombinant Aed
a 1 expressed by the baculovirus system has the same
antigenicity and biological activity as the native Aed
a 1 present in mosquito saliva and is a major salivary
allergen of Ae. aegypti.
EXAMPLE 6
IMMUNOLOGICAL CHARACTERIZATION AND CLINICAL USE OF rAed
a 2, A 37 kDa RECOMBINANT SALIVARY ALLERGEN OF MOSQUITO
AEDES AEGYPTI
Mosquito salivary proteins cause allergic
reactions in humans. As shown in the Examples herein
Aed a 2, a 37 kDa mosquito Aedes aegypti salivary
protein, is an allergen which binds to the IgE of
mosquito-allergic subjects. In this Example, an
expressed, purified recombinant Aed a 2 (rAed a 2), was
characterized to determine if it bound to antibodies
directed to the native protein. Additionally,
responses to it in mosquito-allergic subjects was
investigated to determine its biologic activity, that
is activity in vivo to elicit an immune response.
Sf9 insect cells were co-infected with the
transfer vector pVL1392/Aed a 2 DNA and wild-type
baculovirus. By Western blot using polyclonal rabbit
anti-rAed a 2, the recombinant baculovirus was proved
to express rAed a 2, which was secreted into the
culture medium as a non-fusion protein. The optimal
expression of rAed a 2 occurred at 96 hours after
infection. rAed a 2 was then purified from the culture
medium to homogeneity using anion-exchange (DEAE
Sephacel) chromatography. The rAed 2 was able to bind--
to the IgE of mosquito-allergic sera in Western blot
CA 02309387 2000-OS-12
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and ELISA. This binding was inhibited by addition of
mosquito head and thorax extract in a dose-dependent
manner, showing that the binding of mosquito antigens
to human IgE is specific (Figure 7).
Skin (epicutaneous) tests with purified rAed a 2
and a commercial crude Ae. aegypti extract were
performed in 31 subjects with positive reactions in Ae.
aegypti bite tests and 17 subjects with negative
reactions in the bite tests. Immediate wheals and
flares were measured 20 minutes after the testing, and
delayed reactions were measured 24 hours later. Three
of 31 mosquito allergic-subjects (10%) had a positive
immediate reaction to rAed a 2, compared to ten for the
commercial extract (31%). In the three subjects with
positive rAed a 2 reactions, one subject (3%) developed
a delayed skin reaction to rAed a 2, versus four
subjects tested with the commercial extract (12%).
None of the subjects with negative reactions to
mosquito bites (0%) showed a positive immediate or
delayed reaction to either of the two mosquito
preparations.
Aed a 2 was also shown to be a species-shared
allergen, being present in the saliva or salivary gland
extracts of 6 Aedes and one Culex species among the 12
species studied (Figure 8).
We conclude that the recombinant Aed a 2 expressed
by the baculovirus system has identical antigenicity
and biological activity with native Aed a 2 present in
mosquito saliva and that Aed a 2 is a common allergen
shared by Aedes genus and other species.
EXAMPLE 7
IMMUNOBLOT ANALYSES OF SALIVARY ALLERGENS AND IgE
RESPONSES TO THE ALLERGENS IN 10 MOSQUITO SPECIES WITH
WORLD-WIDE DISTRIBUTIONS
In this Example, saliva or salivary gland extracts
were prepared as described herein above from ten
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mosquito species including seven species with world-
wide distribution. These species are Ae. aegypti, Ae.
vexans, Ae. albopictus, Ae. togoi, Ae. triseriatus, Cx.
quinquefasciatus, Cx. pipiens, Cx. tarsalis, An.
sinensis and Cs. inornata. Proteins from the mosquito
preparations were separated by SDS-PAGE and then
transferred to nitrocellulose membranes. The membranes
were immunoblotted by sequential incubations of the
membranes with human serum, monoclonal anti-human IgE,
and enzyme-conjugated goat anti-mouse IgG. Salivary
allergens were analyzed using a pooled serum from
mosquito-allergic subjects.
Individual IgE responses to each allergens were
evaluated in 12 mosquito-allergic subjects living in
Canada, the USA, and China, as well as in five subjects
who were not allergic to mosquito bites.
Immunoblotting was also performed using two rabbit
antibodies specific to recombinant mosquito salivary
proteins, in order to study species-shared allergens.
Three to 16 salivary allergens with molecular
masses ranging from 16 to 95 kDa were found in each
species (Figure 9). Both species-shared and species-
specific allergens were identified by molecular masses,
binding to the two rabbit antibodies, and the
individual IgE responses to species which were not
indigenous to the areas where the subjects lived.
As shown in Figure 10, the existence of species-
shared allergens was confirmed by immunoblot using
rabbit anti-Aed a 1 and Aed a 2 antibodies,
respectively. A 68 kDa allergen which was recognized
by rabbit anti-Aed a 1 was found not only in the Ae.
aegypti extract (Fig. l0A strip #1) but also in the
extracts of Ae. vexans and Ae. albopictus (Fig. l0A
strips #2 and #3), but not in the extract of Cx.
quinquefasciatus (Fig. l0A strip #4). There were no
allergen bands found in the PBS control strips (data
not shown). Similar results were obtained when rabbit
anti-Aed a 2 antibody was used. A 37 to 39 kDa protein
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recognized by rabbit anti-Aed a 2 was identified in all
3 Aedes species studied (Fig. lOB strips #1 - #3), and
in Cx. quinquefasciatus as well (Fig. lOB strip #4).
Using the sera from individual subjects who lived
in various areas, species-shared and species-specific
allergens were identified, because each mosquito
species is distributed differently. Allergic sera from
Canada (Winnipeg, Manitoba where only Ae. vexans is
found as a major pest) reacted not only with Ae. vexans
but also with the allergens of 5 other species which
are not found in Manitoba. Similarly, sera from China
reacted with the Ae. vexans allergens, a species which
is not present in China. These data show the existence
of species-shared allergens.
Species-specific allergens also existed as
evidenced by a 23 kDa Ae. vexans allergen which reacted
only with the sera from Canada where Ae. vexans is
abundant.
Salivary allergens elicited higher IgE responses
in mosquito-allergic subjects than in non-allergic
subjects. Three major Aedes species (Ae. aegypti, Ae.
vexans, Ae. albopictus) had a higher number of
allergens which also elicited stronger IgE responses,
suggesting that they are major biting species.
During a mosquito bite, the saliva injected may
cause a variety of local and systemic adverse
reactions, for which young children are at high risk.
We report five children age two-four years, evaluated
months after "cellulitis" was diagnosed at the site of
a mosquito bite because of a large, severe reaction
with erythema, edema/induration, and warmth involving
an entire body region (periorbital area, hand, foot, or
leg), for up to two weeks. Systemic symptoms included
fever and irritability. Blood cultures were negative.
The diagnosis was confirmed by measuring Aedes vexans
saliva-specific IgE, IgG, and IgG4 in serum. We also
report control children age two-four years with typical
reactions to mosquito bites, and mosquito bite-negative
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adults.
ELISAs Skeeter child adult
(mean) Syndrome controls controls
n=5 n=5 n=10
IgE (U/mL) 1,491 21 13
IgG (U/mL) 277 9 15
IgG4 (U/mL) 7,174 15 14
In Western blotting, sera from children with
Skeeter Syndrome reacted with 8-15 Aedes vexans
salivary antigens. Skin tests with commercial
extracts, which contain little mosquito salivary
antigens, (Ann Allergy Asthma Immunol 1996; 7:371-6)
were not performed.
Throughout this application, various publications,
including United States patents, are referenced by
author and year and patents by number. Full citations
for the publications are listed below. The disclosures
of these publications and patents in their entireties
are hereby incorporated by reference into this
application in order to more fully describe the state
of the art to which this invention pertains.
The invention has been described in an
illustrative manner, and it is to be understood that
the terminology which has been used is intended to be
in the nature of words of description rather than of
limitation.
Obviously, many modifications and variations of
the present invention are possible in light of the
above teachings. It is, therefore, to be understood
that within the scope of the appended claims, the
invention may be practiced otherwise than as
specifically described.
49
CA 02309387 2000-OS-12
_ WO 99/25826 PCT/IB98/01961
TABLE 1
8 $
_ _
H G L A ~ ~~' W WA ' W0.
W
U . O O O O
7 ~ ~ V O ~ .b
m T .~ . b p, O. P. p"'b p.
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a
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C Z. ~ ~ ~ ~ O vD ~'~ O
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~'.
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a :~ ~ ~ v
a
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x x~
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SUBSTITUTE SHEET (RULE 26)
CA 02309387 2000-OS-12
_ WO 99/25826 PCT/IB98/01961
TABLE 2
b
a0., ~ p ~ ~t M ~ ~Y vWD M
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O
O ~ cd
O ~.,
'cY ~D ~-~ ~ ~O v0 M N ~ ~ O
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o
E
0
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n ~ O ~ O ~ O ~ O O O O O
p, O ~ O O O O O O O O
~U C~ Gl~ M~N~OMO M~"N~D M
N G
O
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O cd ~ N ~ ~ ~ ~ .~~ ~ n
VO O O p vp O O O O
O ~ N V1 V1 V1 y~ O y~ O O O h
3 ~' ~ ° °-° p M c ~ ~ .°° Q,' o°o
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ar
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G1, ~ ~ M N ~~ ~ O d' ~-~ ~ --~ O O O
b a
O
b
O
.O O
O '7
O Tf O O O O O O
O .'! .-, ~ N .~ ~ O
p .a .--, .-n .-r ~ .-, .--n ~ ~ ' in
U ~ ~~r'r~r~~~
.~ g ~ 3 ~~ ,~ .~3 .~ ~ ,~ .o .d ~ ~ .a v
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b ~ ~ 3 ~3 of ~ '~b o3
f* ~U~~~C~C~U ~~~ ~ ~ C
51
SUBSTITUTE SHEET (RULE 26)
CA 02309387 2000-OS-12
WO 99/25826 PCT/IB98/01961
TABLE 3
Skin bite tests and serum mosquito (Ae. vexans) salivary gland-specific IgE
and IgG levels of the
subjects used in this study
SubjectsSkin bite (cm~) Mosquito specific-IgEMosquito specific-IgG
test
No Immediate Delayed(U/ml) (U/ml)
wheat papule
1 I.5 11.6 1026 576
2 1.0 9.6 4341 3285
_ 2,0 7.2 1067 1054
3
4 0.0 0 277 140
0.1 0 112 80
52
CA 02309387 2000-OS-12
- WO 99/25826 PCT/IB98/01961
TABLE 4.
Molecular masses of mosquito antigens in Ae. vexans, Cx. tarsalis and Cs.
inornata (kDa).
Ae. vexans Cx. Cs.
tarsalis inornata
Binding to Binding Binding
to to
IgE IgG IgE IgG IgE IgG
160 160 160 160 160 160
110 110 110 110 110 110
- - 85 85 85 85
80 80 - - - -
- - 70 70 - 70
65 65 65 - 65 -
62 62 62 62 62 62
- - 56 56 56 56
50 SO 50 50 50 50
46 46 46 46 46 46
43 43 43 43 - -
40 40 40 40 40 40
- - 37 37 37 37
32.5 32.5 32.5 32.5 32.5 32.5
- - - - 30 30
- - - 28 - 28
- - - 25 - -
24 24 24 24 24 24
- - 22 22 - -
18.5 18.5 - - - -
Total number 12 12 I S 16 13 14
of antigens
53
CA 02309387 2000-OS-12
- WO 99/25826 PCT/IB98/01961
TABLE 5
GA CCG CCCTTG GTTAAATTA TTG CTA 41
ATT AAA TTC
ATG
AAA
M K P L V K L F L L
TTCTGTCTG GGC ATTGTGCTT TCCAGGCCC ATGCCC GAA 83
GTA
F C L V G I V L S R P M P E
GATGAAGAA GTA GCGGAGGGA GGTGACGAA GAAACG ACC 125
CCA
D E E P V A E G G D E E T ' T
GATGATGCT GGT GATGGCGGC GAAGAAGAA AATGAA GGT 167
GGA
D D A G G D G G E E E N E G
GAAGAGCAT GGA GATGAGGAT GCTGGCGGT GAAGAT ACT 209
GCT
E E H A G D E D A G G E D T
GGCAAAGAG AAT ACAGGACAT GAGGATGCT GGTGAG GAA 251
GAG
G K E E N T G H E D A G E E
GATGCTGGT GAA GATGCTGGC.GAAGAAGAT GCTGAA AAA 293
GAG
D A G E E D A G E E D A E K
GAGGAAGGA AAG GAAGACGCC GGAGATGAT GCCGGA AGT 335
GAA
E E G E K E D A G D D A G S
GATGATGGG GAG GATAGTACA GGAGGTGAC GAAGGA GAA 377
GAA
D D G E E D S T G G D E G E
GCTAACGCT GAC AGTAAAGGT AGTGAAAAG AACGAT CCG 419
GAA
A N A E D S K G S E K N D P
GCCGATACA AGA CAGGTGGTT GCATTACTC GACAAG GAT 461
TAT
A D T Y R Q V V A L L D K D
ACCAAGGTG CAC ATCCAGAGT GAGTACCTT CGATCA GCA 503
GAT
T K V D H I Q S E Y L R S A
CTGAACAAC TTA CAATCAGAA GTGAGAGTT CCGGTG GTG 545
GAT
L N N D L Q S E V R V P V V
GAAGCTATC AGG ATTGGAGAC TATTCCAAG ATTCAA GGA 587
GGG
E A I G R I G D Y S K I Q G
TGCTTCAAA ATG GGTAAAGAT GTAAAGAAA GTTATC AGC 629
TCG
C F K S M G K D V K K V I S
GAAGAGGAG AAA TTTAAGAGC TGCATGAGT AAGAAG AAA 671
AAG
E E E K K F K S C M S K K K
AGCGAGTAT TGC TCGGAGGAC AGTTTTGCG GCTGCC AAG 713
~ ~ CAG
S E Y Q C S E D S F A A A K
AGCAAACTT CCA ATAACCTCT AAGATTAAA TCCTGT GTT 755
TCG
S K L S P I T S K I K S C V
TCATCCAAA CGT TAATGTTAT CATAGTAAG CCATGA ATT 797
GGA
S S K G R Z
TCGATTTGA AAT CCTCATTCT GTCTGTAAC GTTAAT CAT 839
ATA
AAA AAA AAA AAA AAA AAG GAA TTC 863
54
CA 02309387 2000-OS-12
- WO 99/Z5826 PCT/iB98/01961
TABLE 5 (continued)
Primary nucleotide sequence of the Aed a 3 cDNA and its
deduced amino acid sequence. The clone was sequenced
several times in both directions by the Sanger (1977)
method using a US Biochemicals sequence kit. The
putative secretory signal peptide is underlined. A
translation initiation codon (ATG) and consensus
polyadenylation signal sequence (AATAAA) are shown in
bold. The sequence data have been deposited in the
GenBank databases under accession No. AF001927.
CA 02309387 2000-OS-12
WO 99/25826 PCT/IB98/01961
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Racioppi and Spielman, (1987} Secretory proteins from the
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CA 02309387 2000-OS-12
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Peng, Zhikang
Simons, F. Estelle R.
Kohn, Kenneth I.
(ii) TITLE OF INVENTION: RECOMBINANT MOSQUITO SALIVARY ALLERGENS
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kohn & Associates
(B) STREET: 30500 Northwestern Hwy. Suite 410
(C) CITY: Farmington Hills
(D) STATE: Michigan
(E) COUNTRY: US
(F) ZIP: 48334
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Montgomery, Ilene N.
(B) REGISTRATION NUMBER: 38,972
(C) REFERENCE/DOCKET NUMBER: 2595.00027
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 248-539-5050
(B) TELEFAX: 248-539-5055
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 863~base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aedes aegypti
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GAATTCCGAA AATGAAACCC TTGGTTAAAT TATTCTTGCT ATTCTGTCTG GTAGGCATTG 60 .
TGCTTTCCAG GCCCATGCCC GAAGATGAAG AACCAGTAGC GGAGGGAGGT GACGAAGAAA 120
1
SUBSTITUTE SHEET (RUSE 26)
CA 02309387 2000-OS-12
WO 99!25826 PGT/IB98/01961
CGACCGATGA TGCTGGAGGT GATGGCGGCG AAGAAGAAAA TGAAGGTGAA 180
GAGCATGCTG
GAGATGAGGA TGCTGGCGGT GAAGATACTG GCAAAGAGGA GAATACAGGA 240
CATGAGGATG
CTGGTGAGGA AGATGCTGGT GAGGAAGATG CTGGCGAAGA AGATGCTGAA 300
AAAGAGGAAG
GAGAAAAGGA AGACGCCGGA GATGATGCCG GAAGTGATGA TGGGGAAGAG 360
GATAGTACAG
GAGGTGACGA AGGAGAAGCT AACGCTGAAG ACAGTAAAGG TAGTGAAAAG 420
AACGATCCGG
CCGATACATA TAGACAGGTG GTTGCATTAC TCGACAAGGA TACCAAGGTG 480
GATCACATCC
AGAGTGAGTA CCTTCGATCA GCACTGAACA ACGATTTACA ATCAGAAGTG 540
AGAGTTCCGG
TGGTGGAAGC TATCGGGAGG ATTGGAGACT ATTCCAAGAT TCAAGGATGC 600
TTCAAATCGA
TGGGTAAAGA TGTAAAGAAA GTTATCAGCG AAGAGGAGAA GAAATTTAAG 660
AGCTGCATGA
GTAAGAAGAA AAGCGAGTAT CAGTGCTCGG AGGACAGTTT TGCGGCTGCC 720
AAGAGCAAAC
TTTCGCCAAT AACCTCTAAG ATTAAATCCT GTGTTTCATC CAAAGGACGT 780
TAATGTTATC
ATAGTAAGCC ATGAATTTCG ATTTGAATAA ATCCTCATTC TGTCTGTAAC 840
GTTAATCATA
AAA.'9AAAAAA AAAAAAGGAA TTC 863
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /deac = "Primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
GACTCCTGGA GCCCG 15
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /dear = "Primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
ATATCTGTCC ACCAACG 17
2
SUBSTITUTE SHEET (RIJ~.E 26)
