Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~VO92/13~60 PC~/US9~/~&~
~10~788
REAGENTS AND METHODS
FOR IDENTIFICATTON OF VACCINES
Technica~ Field
The invention is directed to reagents and
methods to identify and screen candidates for vaccines.
The identification and screen depends on the capacity of
biological materials used as the screening reagents to
destroy or impair the infective agent in an ln vivo
incubator. More particularly, the invention concerns the
u~e of cells, serum or fractions thereof obtained from
exposed natural hosts wherein a recoverable implant of
infectious agent is used to assess the protective effect
when these materials are provided passively to the animal
incubator~ The method is illustratively applied to
determine useful active agents for an anti-heartworm
vaccine.
Background Art
The general method provided by the invention
below to obtain suitable immunogens for use in vaccines
is applied specifically to heartworm infection in canines
and other mammals, which is caused by the nematode
Dirofilaria immitls. Accordingly, a preliminary
discussion of the nature of this infection and the life
cycle of the D. immitis parasite may be helpful both in
reviewing the background literature and in describing the
invention. The adult forms of the parasite are quite
large (males are 12-20 cm long and 0.7-Q.~ mm wide;
females are 25-31 cm long and 1.0-1.3 mm wide) and these
preferentially inhabit the heart and pulmonary arteries
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of the dog. The sexually mature adults, after ma~ing,
produce as embryos microfilariae which are only 300 ~m
long and 7 ~m wide. These traverse capillary beds and
circulate in the vascular system of the dog in
concentrations of 103-105 microfilariae per ml of blood.
one way of demonstrating infection in the dog is to
detect the circulating microfilariae.
If the dog is maintained in an insect-free
environment, the life cycle of the parasite cannot
progress. However, when microfilariae are ingested by
the female mosquito during blcod feeding on an infected
dog, subsequent development (but not, of course, increase
in numbers) occurs in the mosquito. The microfilariae go
through two larval stages (Ll and L2) and finally become
lS mature third stage larvae (L3) of about 1.1 mm length,
which can then be transmitted back ~o the dog through the
bite of the mosquito. It is this L3 stage, therefore,
that accounts for the initial infection. As early as
three days after infection, the L3 molt to the fourth
larval (L4) stage, and subsequently to the fifth stage,
or immature adults. The immature adults migrate to the
heart and pulmonary arteries, where they mature and
reproduce, thus producing the microfilariae in the blood.
"Occult" infection with heartworm in dogs is defined as
that wherein no microfilaremia is demonstrable, but the
existence of the adult heartworms can be determined
through thoracic examination.
Control of heartworm infection in canir.es has
largely been chemoprophylactic, and no effective vaccine
is available for practical immunization of the dog
population against this parasite. Further, there appears
no generic method to determine suitable immunogens for
use as active ingredients in vaccines directed against
infectious diseases caused by parasites in general. The
invention provides solutions to both of these problems.
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As to t~e general approach to a method to
obtain vaccine components, the ability of various
components of infectious agents to raise antibodies when
injected into animal hosts is well understood not to be
determinative of the ability of these components to
behave as e~fective vaccines. A large number of
materials are immunogenic and produce sera which test
positive in immunoassays for ability to react with the
immunizing antigen, but which fail to protect the hosts
against infection. Antibodies which neutralize the
infective agent in in vitro assays are much more likely
to protect against challenge in v~vo. Accordingly, the
use of "immune" serum simply resulting from
"immunization" or from infection by the infectious agent
to screen for candidate vaccines does not provide
sufficient specificity to identify protective immunogens.
on the other hand, serum or other components of blood
from immunized animals which is demonstrably protective
against infection is assured to contain antibodies,
cells, or other fastors reactive with an immunogen or
infectious agent which will produce responses that
protect against challenge.
In most infectious diseases, particularly those
such as parasitic infections that have long and complex
development courses, it is difficult to verify the
protective effect of serum or T-cells from exposed
animals for use as a screening reagent. First,
verification of protection against challenge is tedious,
since the host animal would first have to be challenged
with the infectious agent and shown to be protected
before it could be shown that antibody components of
serum, for example, could be used as a screen. The
definition of "protection" under such a regimen is often
complex. Second, even if a protective effect against
challenge is shown, it is not clear to what components of
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the immune system the protection is due. The protective
effect could ~e due to antibodies, cells, mediators of
the immune system or to combinations thereof. Thus,
although this method of obtaining the screening reagent
is sometimes used, it is time-consuming and does not
permit identification of protective components.
A more useful manner of obtaining blood
components and substantiating their protective effects
takes advantage of implanted diffusion chambers
containing the infectious agents, such as those described
by Grieve, R.B., et al. Am J Trop_Med Hyg (1988) 39O373-
379, and by Abraham, D., et al., J Parasit (1988) 74:275-
282. In the first of these papers, dogs which had been
immunized against Dirof1laria immitis infection were
supplied diffusicn chambers containing infective larvae.
The larvae in the chambers could then be evaluated for
the effect of the previous immunizations. In the second
paper referred to, mice were supplied diffusion chambers
containing D. immitis third-stage larvae, and the effects
on these larvae were used to determine the possible
immunity of the mice putatively engendered by previous
injections with L3. In the context of these papers, the
use of the diffusion chamber containing the infectious
agent, therefore, gives a convenient assessment of the
effectiveness of certain directly administered active
imm~nization protocols, but not of passively transferred
protective effects of selected fractions of a ~arget host
bloodstream.
The general method of the invention is
illustrated specifically below in the context of
heartworm infectio~ in dogs, a disease which is
identified as one wherein the complexity of the
parasitic infection makes the choice of a candidate
immunogen for vaccination very difficult. Even naturally
conferred immunity cannot be assured to exist, as dogs
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with previous or existing infections with D. immitls can
be reinfected (Grieve, R.B., et al. E~idemiolo~ic Reviews
(1983) 5:220-246). However, this review also reports
that there is some evidence of a naturally occurring
protective immune response. This evidence is stated to
be the apparent limitation on the population of mature
worms in infected dogs.
Furthermore, it has been possible to induce
protective immunity artificially. Wong, M.M., et al.,
Ex~ Parasitol (1974) 35:465-474, reported the
immunization of dogs with radiation-attenuated infective
larvae. The dogs were protected to varying degrees upon
challenge. Blair, L.S., et al., in Fifth International
Conaress of Parasitology, Toronto, Canada (August 1982),
reported successful immunization by infecting the dogs
and terminating the infection at the fourth larval stage
by chemotherapy.
Grieve, R.B., Proc Heartworm S~m~ (1989), pp.
187-190, reviewed t~e status of attempts to produce
vaccines against heartworm in dogs. This report
summarizes the use of infective larvae implanted in an
inert diffusion chamber which permits the influx of cells
and/or serum from the host and outflow of parasite
material from the chamber to assess the effectiveness of
inoculation protocols in both dogs and mice. The use of
immunization with infective larvae was demonstrated to be
partially effective in protection against subsequent
challenge.
An alternative approach to finding a heartworm
vaccine has been to attempt to identify prominent
antigens in the infective stage of D. immitis. Philipp,
M., et al., J Immunol (1986) l36:2621_2627, reports a 35
kd major surface antigen of D. immitis third stage larvae
which was capable of immunoprecipitation with sera from
doss carrying an occult experimental D. immitis infection
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or with sera fro~ dogs immunized by irradiated third
stage larvae. In addition, this group reported (Davis,
T.B., et al., Abstract 404, 37th Annual Meeting, Am. Soc.
Trop. Med. Hyg. (1988)) three major surface proteins of
the L4 stage of molecular weights 150 kd, 52 kd, and 25
kd. The 25 kd molecule seemed unique tc the L4 stage.
Ibrahim, M.S., e~ al., Parasitol (19~9) 99:89-
97, using D. immi~ls L3s labeled with l25I, showed that a
35 kd and 6 kd component were shed into the culture
medium by developing parasites. They further showed that
antibodies from immunized rabbits and infected doqs
immunoprecipitated the 35 kd, but not the 6 kd,
component.
Scott, A.L., et al, Acta Tro~ica (1990) 47:339-
353, reported characterization of the surface-associated
molecules of L2, L3, and L4 of D. immitis by
radiola-beling techniques and SDS-P~GE. They found major
labeled components of 35 kd and 6 kd in extracts from
iodine-labeled L2 and L3 stages; lactoperoxidase~
catalyzed labeling revealed Components Of apparent
molecular weights 66 kd, 48 kd, 25 kd, 16.5 kd, and 12
kd. Iodine labeling of surface-associated molecules of
L4 gave molecules of apparent molecular weights of 57 kd,
40 kd, 25 kd, 12 kd, and 10 kd; lactoperoxidase-catalyzed
labeling showed additional bands of 45 kd, 43 kd, and 3
kd.- However, these were identified using uncharacterized
serum sources or without serum identification.
Other approaches to obtaining vaccines against
parasites in general have focused on the production of
neutralizing antibodies. For example, bo~h in vitro
studies by Tanner, M., et al., Trans Roy Soc Trop Med HYG
(1981) 75:173-174, and by ~im, B.K.L. et al., (ibid.)
(1982) 76:362-370, and in vivo by Parab, P.B., et al.,
Immunol (1988~ 64:169-174, have demonstrated that anti-
bodies are effective alone or with other immune
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components in killing filarial L3 from Di~etalonema
(Acanthocheilonema) viteae or Bruqia malayi.
Furthermore, passive immunity to Schistosoma mansoni has
been transferred from immune rats or humans to normal
mice (Sher, A. et al., Parasitol (1975) 70:347-357; Jwo,
J. et al., Am J Trop Med Hy~ (1989) ~l:553~562). None of
these studies involved recovery and evaluation of the
infectious agent implanted in an in vivo incubator for
the agent treated with candidate protective components.
Disclosure of the Invention
The invention provides both a general
methodology for identification of suitable immunogens for
inclusion in vaccines and, specifically, immunogens
identifi~d using this method which are useful in
controlling heartworm infections in dogs. The general
methodology depends on the verification of the protective
qualities of serum or cells used to detect or bind
candidate antigens by virtue of the ability of the serum
or cells to impair or destroy thè infectious agent in an
in vivo incubator animal wherein an irrelevant host
contains diffusion chamber implants of the infectious
agent.
Accordingly, in one aspect, the invention is
directed to a reaqent useful to identify immunogens for
inclusion in vaccines against infectious agents. The
reagent comprises cells, antiserum or fractions thereof
-which has been demonstrated to be protective against the
infective agent by its ability to confer, destroy or
impair the infectious agent in an in vlvo model wherein
an irrelevant host is implanted with a diffusion chamber
containing the infectious agent. The method is, of
course, limited to infectious agents of sufficient size
to be retained by the diffusion chamber.
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In another aspect, the invention is directed to
methods to use these reaqents to screen for candidate
vaccines in various potential sources of immunogens.
These sources of immunogens may be extracts or resolved
extracts of the infectious agent, or DNA expression
libraries obtained from the infectious agents. The
invention is also directed to methods to confer passive
immunity on hosts by administration of the reagent.
In other aspects, the invention is directed to
; lO the application of the reagents and methods of the
invention to heartworm infection and, specifically, to a
component of the L3 and L4 larval stage of D. immitis,
said component having a molecular weight of 39 kd.
Additional components verified to react exclusively with
protective immune serum cells or fractions are also
disclosed.
In still another aspect, the invention permits
evaluation of the candidate immunogens by providing a
short-term test of their protective effect in the target
host. Instead of challenging the immunized hosts with
infectious agents directly and waiting several months to
evaluate the outcome of infection, the host may be bled
and the components of the blood or serum tested more
directly in the experimental host containing the
2S diffusion chamber. This permits rapid screening of both
naturally occurring components of the infectious agent
and of synthetic peptides, carbohydrates and glyco-
proteins.
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Brief Description of _he Dr~
Figure l shows Western blots of D. immitis
proteins immunoreacted with canine sera derived from
immune and nonimmune dogs.
5Figure 2 shows Western blots of D immitis
proteins immunoreacted with canine sera at various time
points (days) after immunization.
Figure 3 shows the results of SDS-PAGE on
proteins labeled with S-35 methionine extracted from D.
immitis L4 larvae and reacted with control and immune
sera at various time points after immunization.
Figure 4 shows the results of proteins analyzed
as set forth in Figure 3, but wherein the larval surface
proteins are labeled with I-125.
Figure 5 shows the results of proteins analyzed
as in Figure 3, but wherein the larval surface proteins
are labeled using biotin.
Figure 6 shows the results of analysis of
; proteins present in the~excretory/secretory material
which characterizes the transition from L3 to L4 and
maintenance of L4's for 3-4 days thereafter. s
Modes oP Carrvinq Out the Invention
The method of the invention takes advantage of
a model system which has been used to evaluate
immunization protocols using the active response of the
model host, but not to ascertain or validate the
protective capacity of specific immune reagents generated
in the natural target host and passively transferred into
the experimental animal system. Thus, the method has not
been used to identify suitable reagents which themselves
are useful to _dentify and evaluate vaccine candidates.
The active immunization model as applied to heartworm is
described in the 1988 papers of Grieve and Abraham cited
above. In this model, the infectious a~ent, third-stage
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larvae, were first obtained as follows. D. immitis was
maintained in a dog infected with parasites which had
been passed once from an infection obtained through the
U.S.-Japan Cooperative Medical Sciences Program, National
Institutes of Health. Aedes aegypti mosquitos Liverpool
tblackeye strain) were infected with the parasite by
feeding on microfilaremic blood using the artificial
feeding apparatus described by Rutledge, L.L., et al.,
Mosq News (1964) 24:407-419. Fifteen days after
infection, the mosquitoes were cold-anesthetized and
surface sterilized by immersion in 95% ethanol followed
by a 3 min wash in 1% benzalkonium chloride in 0.01 M
phosphate-buffered saline, pH 7.2. The mosquitoes were
washed three times in PBS and incubated over a 60 mesh
screen inside a funnel filled with medium (the medium was
described in Abraham, D. et al., J Parasitol (1987)
`~ 73:377-383) and the larvae were collected 90 min after
incubation.
The recovered L3 larvae are then placed in
diffusion chambers and implanted subcutaneously. For
implantation in dogs, chambers are composed of two 14 mm
Lucite rings sealed with S.0 ~m hydrophilic Durapore
membranes (Millipore, Bedford, MA). The larvae are
inserted through a hole in one of the Lucite rings which
; 25 is subsequently sealed with nylon thread. The dogs are
anaesthetized and a subcutaneous pocket is formed in the
dorsal skin of the neck; the chamber is implanted in the
pocket and the wound closed with suture.
For implantation in mice, similar chambers are
used and implanted into a subcutaneous pocket formed
laterally to the lumbar spine. The chambers can be
removed at the desired time for evaluation of the
contained larvae.
A variety of chamber desiqns can be used, and
3S the porosity of the diffusion membrane chosen according
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to the nature of the infectious agent and ~o desired
llmitation on the nature of the inward diffusion.
In the use of the model to prepare screening
reagents or to evaluate samples from target animal hosts,
the diffusion chambers are allowed to remain in vivo in
an irrelevant host which has not been actively immunized
and a portion of serum or other component believed to be
capable of conferring protective immunity is administered
to the irrelevant host. A portion is retained for use in
the invention method to screen immunogens if destruction
or impairment of infectious agent is shown. For use in
dogs as a model, for example, about 0.5 ml of serum, for
example, is administered by placing the serum in the
pocket along with the diffusion chamber. For mice,
similar amounts but fewer chambers are used. The chamber
containing the infectious agent is allowed to remain in
place for a time sufficient to evaluate the protective
effects of the serum or other component. This time is
determined by the nature of the infection and the
protective capacity of the test sample.
After the required experimental time has
lapsed, the chamber is removed and the infectious agents
are retrieved. The protective capacity of the passively
transferred components from the natural target host is
evaluated by any deleterious effects seen in the
infectious agents. These effects may include, but are
not limited to, such parameters as killing, stunting,
alterations in normal morphology, alterations in
measurable metabolism, failure to mature in in vitro
culture, and failure to infect conventional target hosts.
A fraction of the sample which has been
retained during the evaluation is then used to screen
candidate immunogens. Any technique which results in the
complexation of the validated component with the
candidate immunogen can be used. For example, an extract
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of the infectious agent is subjected to resolution using
a variety of chromatographic techniques, including size
separation using gel permeation chromatography,
electrophoresis on polyacrylamide gels, ion exchange
chromatography, affinity chromatography, and the like.
The whole extract or resolved extract is then tested for
reactive effect with the protective component. If serum
is the protective component, a complex will be formed.
If the component is a cell subfraction with receptor for
antigen, the antigen will be bound. The complex is
recovered, and the immunogen recovered from the complex.
In applying the method to crude extracts, the
protective serum or cells can be used as an affinity
ligand in chromatographic techniques to isolate
immunoreactive components. Alternatively, as set forth
above, the extract can first be resolved and the
appropriate fractions identified by complexation with the
protective cells or serum.
In an alternative approach, the protective
cells or serum can be used as a screening reagent for a
cDNA library prepared from the infectious stage or later
stage of the infective agen~ which is constructed in
expression vectors. A commonly used and convenient such
library is the Agtll library described by Young, R.A.,
and Davis, R.W., Proc Natl Acad sci !USA) (1983) 80:1194-
1198. The expression library is platecl and screened with
the protective cells or serum to identify colonies which
produce immunoreactive components. The positive colonies
are then purified, and the cDNA inserts in the expression
vectors recovered and sequenced to identify the encoded
proteins.
The cDNA inserts identified as expressing
immunogens using the reagent of the invention can then be
ligated into alternative conventional expression systems
for production of the proteins useful as vaccines.
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Alternatively, the inserts may be ligated into expression
systems which are live recombinant carriers such as
Sindbis virus, vaccinia virus or other pox viruses,
Herpes viruses, Adenoviruses, Salmonella or
Mycrobacteria. These infectious agents can then be used
directly to immunize the target hosts by generation of
immunogen in situ.
The vaccines of the invention are administered
in a manner consistent with the nature of the vaccine and
the nature of the disease and subject. If the
recombinantly produced or native immunogens are
administered as proteins, they are formulated with
conventional excipients for injection or other systemic
administration to the host. In addition to injection,
lS formulations may be prepared for other administration
methods which include transmucosal or transdermal
delivery into the bloodstream. If the immunogens are
administered in the form of recombinant DNA expression
systems in infectious agents, administration is typically
by injection or other mode of conventionally
administering the infective agent.
The foregoing approach is applicable to the
discovery of suitable immunogens for any disease wherein
the infectious agents can conveniently be used in the
animal model. In general, such diseases are those caused
by organisms of sufficient size to be retained in a
diffusion chamber. Thus, in general, the method is
applicable to a variety of parasitic diseases including
those caused by other filarial nematodes, such as
Di~etalonema ~erstans, DiPetalonema stre~tocerca,
Wuchereria bancrofti, B. malavi, Mansonella o7zardi, Loa
loa~ and o. volvulus.
Other disease-causing oryanisms to which the
method is applicable include Stronqyloides spp.,
Stronaylus spp., Haemonchus spp., Trichostronqvlus spp.,
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ostertaqia spp., Coo~eria spp., Dictyocaulus spp.,
Nematodirus spp., Cyathostominae (small strongyles of
horses), Oesophaqostomum spp., Chabertia ovina,
Ancylostoma spp., Uncinaria spp., Bunostomum spp.,
Filaroldes spp., Aelurostronqylus abstrusus, those
nematodes of the Order Ascaridida (Ascarids), Trichinella
spiralis, Trichuris spp., Angiostrongylus spp.,
Enterobius vermicularis. In applying the method, the
infectious stage of the agent is determined, if
applicable, for identification of the form to be enclosed
in the chamber. Protocols are adjusted to take account
of the time required for exertion of the prctective
ef~ect of the target animal-derived component, such as
cells or serum.
Using the invention reagent, several immunogens
associated with D. immitis were obtained. One class of
such immunogens have molecular weights of 66 kd, 65 kd,
59 kd, 39 kd, 33 kd, 23l24 kd, 22/20.5 ~d and 14 kd. The
foregoing proteins are produced by L3 and/or L4 larvae.
DNAs encoding these proteins can be recovered from cDNA
expression libraries prepared from the mRNA of these
larval stages by screening for expression with the
reagent of the invention. The appropriate cDNA inserts
can then be used to produce recombinant immunogen in a
suitable expression system which yields practical amounts
of the protein or can be ligated into recombinant systems
which generate the immunogen in sltu, such as the
vaccinia virus system.
The following examples are intended to illus-
trate, but not to limit the invention.
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Example 1
Production_of_Sera for Passlve Transfer
Four dogs were immunized with chemically-
abbreviated infections, and two dogs served as
chemically-treated controls. The dogs were housed in
indoor mosquito-free individual cages at a temperature of
22C and 40-65% humidity. On day 532, post initial
immunization, each dog was challenged with 100 L3
D. immitis larvae contained in 5 diffusion chambers,
described above. Concomitant with chamber implantation,
the dogs were injected subcutaneously with 50 L3 and the
infection was allowed to proceed beyond the anticipated
pre-patent period. Challenge in~ections were repeated on
day 588 with 100 larvae within diffusion chambers and 30
L3 inoculated subcutaneously. Serum was collected at
numerous time points from the immunized dogs, including
554, 588, 602 and 642 days after initial immunization
whi~h corresponded to days 22, 56, 77 and 117 after
initial challenge. The isolation of serum provides a
source of immunoglobulins and soluble factors, but not of
cells. Antibody levels were measured to L3 and L4
surf~ce antigens using an indirect fluorescent antibody
assay and to L3 and L4 soluble antigens and an excretory-
secretory antigen fraction by an indirect ELISA, as
dPscribed by Grieve et al., ~19~8) (supra). The sera
were pooled and validated as a significant factor in the
protective effect in the mouse incubator as described in
Example 2.
Exam~le 2
Validation in Mouse
A subcutaneous pocket was formed in male Balb/C
BYJ mice approximately 10 weeks old and 20 L3 inoculated
into diffusion chambers as described above were implanted
into the pocket, along with 0.5 ml of the demonstrated
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w092~l3s60 PCT/VS92/~0~
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protective serum to be tPsted. Serum samples were
retained for future use. The diffuslon chambers werP re
covered two or three weeks later. Living larvae in the .
chambers were counted and placed into glacial ace~ic acid
followed by 70% ethanol con~aining 5% glycerin. The
ethanol was allowed to evaporate leaving the larvae in
glycerin; the larvae were measured using projected images
in the Macmeasure image analysis system on a Macintosh
computer.
Three groups were used: experiment 1 used
equal portions of serum from individual dogs a~ each of
the three collection points described in Example 1 (days
56, 77 and 117). In experiments 2 and 3 only sera from
immune dogs 117 days after initial challenge were used.
Control sera were used in all cases; in experiment 2 this
was a pool from 12 naive dogs; in experiment 3 from an
individual dog. These groups also contained controls
which received no serum.
In experiment 1, chambers were recovered two
weeks post-inoculation. The survival rate of larvae in
chambers from mice receiving serum from immune dogs were
lower than those from mice receiving normal dog serum,
but the difference was not statistically significant.
Also, no difference was seen between the length of larvae
in each case.
. In experiments 2 and 3, the chambers were
recovered three weeks after infection. There were
significant differences în the larval recoveries between
those receiving serum from naive dogs and those from
immune dogs--about 34-33%. The lengths of the larvae
were also significantly shorter in those chambers
receiving sera from immune dogs.
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Example~3
Identification of Antiqens
Crude extracts of L3 and L4 larvae were
prepared as follows:
All procedures are performed at 4C or on ice.
The worms were collected and washed twice with wash
buffer (PBS/0.1% Triton X-100) and then with extraction
buffer (0.05 M Tris/HCl~ pH 6.8; 2% CHAPSO; 1 mM PMSF; 1
mM EDTA; 1 mg/l leupeptin; 1 mg/l pepstatin). (Other
detergents may be used in place of CHAPSO, includiny O.5%
Triton X-100, 0.5% CTAB, 2% DOC, or 2% SDS/5% 2-ME/8M
urea.)
The worms are then homogenized 5x for 1 minute
each, with l minute rest periods, using 250 to 500 ~l for
10,000-20,000 worms (- 500 ~g). This volume i5
transferred to an additional tube, and the homogenizer
washed with a clean 100-250 ~l of extraction buffer and
the wash pooled with the homogenate. The tube is rocked
4 hours-overnight and centrifuged at 12,000g for 10
minutes. The supernate is harvested and the pellet is
washed once with extraction buffer and saved for
additional extractions if desired. The combined total
volume o~ extract is less than 1 ml and about 20 ng of
protein is solubilized per L4 larva used.
The procedure for L3 is identical, except that
the.wash buffer is PBS without detergent.
The extracts were subjected to polyacrylamide
gel electrophoresis and tested with portions of the serum
shown to be protective in the murine model. When pooled
canine sera which had been shown to stunt larval growth
as described in Example 2 were used as the immuno-
reactant in the Western blots, the results were as shown
in Figure 1. The 39 kd band shown in Figure l is
separated from a 45 kd band when a second dimension is
added to the electrophoresis. This 45 kd protein is not
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immunoreactive. As seen, the serum is specifically
immunoreactive with a 39 kd protein present in the L4
larval stage. This protein has a pI of about 5. Control
serum shows no immunoreactivity with this protein.
Reactivity to the 39 kd molecule is present in immune
dogs, but not in control dogs. Sera from dogs with
microfilaremic infection or amicrofilaremic infection do
not recognize this molecule.
In addition, bands were present at 66 kd,
24/23 kd, and 14 kd, as shown in Figure 2.
The proteins associated with the larval stages
were also metabolically labeled using S-35 methionine; or
the surfaces were labeledt prior to extraction, with
I-125 or with biotin. For labeling with S-35 methionine,
the radiolabeled amino acid was added to the parasites
after 48 hrs in culture according to the method of
Abraham, D., et al., J P _asitol (1987) 73:377-383. For
labeling with I-125, the method of Mok, M., et al., Molec
Biochem Parasitol (1988) 31:173-182, was used. For
biotinylation, a modification of the method of Alvarez,
R.~., et al., Molec Biochem Parasitol (1989) 33:183-190,
was employed. In the modified procedure, NHS-longchain
biotin was substituted for biotin per se.
Thus, additional identification could be had
using these prelabeled proteins which immunoprecipitated
with.the successfully validated immune serum. These
results are shown in Figures 3, 4 and 5. As shown in
Figure 3, additional candidates are found at 59 kd and 16
kd, as indicated by the arrows. The radioactive iodine-
labeled material shows a candidate at about 33 kd with ahigher moiecular weight smear at 35.8-34.5 kd. This was
present beginning at day 345 and persisting until day 642
in some, but not all, immune dogs. An additional band
was present at 14.5 kd. This is indicated in Figure 4.
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W~s2/l3560 PCT/US92/~
2~03788
--19--
Figure 5 shows the results when the proteins
were labeled by biotinylation in an enhanced chemi-
luminescence assay. A transient band represented by
65.3 kd was recognized by 3 of 4 immune dogs.
In addition, passive transfer of the earliest
immune dog serum which showed uniform responses to the
39 kd protein (i.e., the day-142 immune serum shown in
Figure 3) was able to effect killing of the entrapped
larvae; recoveries of intact larvae were only S8.3~ in
the case of immune serum compared to 65.8~ for controls.
To summarize, the following antigen candidates
were obtained:
A 39 kd protei~ which reacted with sera from
all immune dogs but not with sera from naive cohorts.
The protein is shown to be present in Western blots
obtained from L4 soluble antigen and solubilized L4
larval pellets and is shown to be present, although
apparently to a lesser degre~, in L3. This protein
appears to be absent from~adult D. immitis and the
microfilariae. It is clearly a distinct protein from the
p35 protein described bv Scott, A.L., et al., Acta
Trop~ (1990) (supra), and is relatively acidic, having
a pI of approximately 5.
A 14 kd immunogen is detected with immune dog
seru~ using Western blots and immunoprecipitation
employing S-35 and iodine-labeled components. The
protein is detected with immune dog serum, but not by
serum from controls.
Additional proteins detected are of 66 kd and
30 23/24 kd.
Another potential source of protective antigens
in parasitic diseases are excretory/secretory products
which are associated with various stages of the parasite.
The transition between L3 and L4 involves excretionl
3S secretion of a number of proteins which are harvested as
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W092/l3560 PCT/US92/00~
2103788 -2
follows: Larvae are cultured at 250-400/ml, washed at
48 hr and cultured an additional 4 days. The worms are
then settled out and the supernate collected. This is
filtered through a 0.45 ~m filter and protease inhibitors
added as in L4 solubilization. The ES is then concen-
trated and buffer exchanged by ultrafiltration over a
10 kd membrane (Amicon Centriprep-10 and/or Centricon-
10). The final buffer is 0.05 M Tris/HCl pH 6.8 with
protease inhibitors. ~ields may be app. 5 ng/larvae.
10 Final volume frequently 150-250 ~1. This extract,
referred to as DILEX, was prepared using larvae which
were metabolically labeled with S-35 methionine and
tasted with respect td immune and control sera from dogs.
The immune serum was that obtained on day 554 post
immunization as set forth in Example 3.
Immunoprecipitation with respect to the immune serum was
- obtained at 22/20.5 kd and 14.3 kd, as shown in Figure 6.
In Figure 6, lane 1 shows molecular weight standards;
lane 2, the immunoprecipitates from immune dog; lane 3,
- 20 from control dog; lane 4, bead control; and lane 5, DILEX
~ itself.
,
Example 4
Isolation of cDNA's and Genes Encodina Potential
D. immitis Protective ImmunQ~ens
. Genomic and cDNA expression libraries in AZapII
(Short, J.M., et al., Nucleic Aclds Res (1988) 16:7583_
7600), a derivative of ~gtll, were prepared from total
genomic DNA, or L4 or L3 larval stage mRNA's,
respectively, using standard procedures (Short Protocols
in Molecular Biolo~y (1989) Ausubel, M.F., et al., eds.)
Screening of these libraries with pooled immune dog sera
permits identification of clones which contain candidate
antigens. The clones identified as immunoreactive with
the immune serum provide a source of DNA encoding desired
WO92~135~ PCT~US92/~0
proteins which can conveniently be produced as fusion
proteins in E. CQli.
Example 5
Construction of GST ~usion Proteins
The DNA inserts are recovered from the AZapII
phaqemid by digesting with EcoRI and purified using
agarose gel electrophoresis. The purified DNA is ligated
into the expression vector pGEX-3X such that when the
plasmid is expressed in E. coli the protein enooded by
the DNA insert produces a fusion protein with
glutathione-S-transferase. This procedure is described
in detail by Smith, D.B., et al., Gene (19g8) 67:31-40.
Plasmids containing the DNA inserts are transformed into
E. coli and successful transformants are grown in the
presence of IPTG. The induced fusion protein is purified
from the lysate by affinity chromatography with
glutathione-beads as described by Smith et al. (supra).
Example 6
Immunization of Doas
Recombinant peptides derived from any of a
variety of expression systems are used to immunize dogs
for the purpose of obtaining specifically reactive blood
components. Recombinant antigens are administered to
dogs with or without adjuvant by the subcutaneous,
intramuscular, intradermal or intravenous routes.
Following single or multiple immunization, blood is
collected from dogs by routine venipuncture. Serum is
collected from coagulated blood and used directly or
stored frozen prior to use. Leukocytes are collected
from anticoagulant-treated blood by density gradient
centrifugation and used directly or stored by freezing at -
1C/minute with storage in liquid nitrogen.
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