Note: Descriptions are shown in the official language in which they were submitted.
2:123~2{)
W~93/10225 PCT/US92/09702
PROTEASE VACCINE AGAINST HEARTWORM
Technical FieId
The invention relates to prevention andtreatment of nematode-caused filarial disease in animal
hosts, such as heartworm which occurs most commonly in
dogs. Heartworm infection is caused by the nematode
Dirofilaria immitis, and the treatment and prevention
method of the invention can be applied specifically to
this disease by employing the characteristic metallo-
protease and/or cysteine protea~e associated with this
organism.
:
Backqround Art
The heartworm i~fection caused by D. immitis is
a widely distributed problem in dogs in most regions ofthe world with the exception of Africa. Current
treatment is generally chemoprophylactic with ~gents
designed to directly kill the infecting organisms. While
this treatment has gained accepta~ce, because of the
; 25 inherent toxici~ty of such treatment, it would be
preferable to immunologically protect the host against
i~fection, or to revise the chemoprophylactic regime to
i~clude less toxic agents. The present in~ention is
directed to this goal.
Other nematode filarial infections are of even
greater significance and in~-olve life cycles of the
infectious agent similar to those related to heartworm.
For example, o~ more concern are the other filarids which
infect humans, and more than 200 million people worldwide
are estimated to have such infections. Filarid~ which
W093/l0225 2 1 2 3 ~ 2 0 -2- PCT/US92/09702t
infect humans include Brugia malayi, Wuchereria
bancrof~i, and Onchocerca volvulus. These are serious
infections which can cause blindness and elephantiasis in
humans. At present, there is no effective vaccine
available against filarial nematode infection.
As the life cycles of the infectious agents are
similar in all of these diseases, heartworm infection can
be used as an illustration. This life cycle can be
described as follows:
Heartworm infection, specifically in dogs,
generally occurs through passage of the third-stage
larvae (L3) of the nematode D. immitis into the
subcutaneous ti~sue from a mosquito vector. When these
larvae are passed into the animal~s tis~ue, their life
cycle is continued by molting into a fourth larval stage
(L4), which then migrates toward the heart and pulmonary
arteries where the subsequent stage matures into an
adult. The ~3 remain at the site of inoculation by the
mosquito until molting occurs. ~4 emigrate through
cutaneous tissue and muscle ~nd do not molt to fifth
stage for 50-70 days af~er infection (Grieve, R., et al.,
Epidem Rev (1983) 5:220-246). Adult D immitis, which
are on the order of 12-20 cm (males) and 25-31 cm
(females) in length, produce, in this location, motile
vermiform embryos called microfilariae, which are o~ly
0.3 mm long and which traverse capillary beds and
circulate in the ~ascular system. The microfilariae are
ingested by mosquitoes and continue their life cycle
through L3 in the mosquito vector.
The transition from L3 to h4 i5 thus the
initial step in the infection cycle in the animal ho~t.
This transition involves a molting process, which has
been studied by Abraham, D., et al., Experimental
Parasitol (1990) 70:314-322, incorporated herein by
reference. In this study, the morphology of the larvae
wO 93/10225 2 ~ ~ 3 ~ 2 0 PCT/US92/09702
-3-
during the molt was monitored, and the roles of
temperature and of albumin, which appeared to be
essential for this process, were evaluated. The L3 stage
contains a cuticle and epicuticle which are abandoned,
and the body wall of the larva is encased in the L4 stage
cuticle and epicuticle. During this molting process, an
excretory-secretory (E-S) product which, among other
components, contains enzymes presumably employed in the
molting proceqs is formed.
The excretory-secretory products of various
tissue invasive helminths have been studied. Proteases
have been found in a number of them. Both Schistosoma
mansoni and Schistosomatium douthitti produce elastases
capable of degrading skin (~cKerrow, J.H., et al.,
Experimental Parasitol (1982) ~3:249; McKerrow, J.H., et
al., J ~iol Chem (1985) 231:47; Amiri, P., et al., Mol
Biochem Parasitol (1988) 28:113). Fasciola hepa~ica also
release~ a number of proteolytic enzymes (Dalton, J.P.,
et al., ol Biochem Parasitol (19~9) 35:161). The adult
hookworm ~ncylos~oma caninum releases a histolytic
protease and a protease that acts as an anticlotting
agent (Hotez, P.J., et al., J Biol Chem (1985) 260:7343).
Toxocara anis larvae secrete proteases which degrade
cQmponents of extracellular matrix (Robertson, B.D., et
al., Bxperimenal ~E~_itol ~1989) ~:30).
A number of filarial nematodes also have been
hown to produce proteases that act on ex~racellular
matrix co~ponents, including Onchocerca cervipedis, O.
cer~icalis, and Bruqia malayi (Lackey, A., et al.,
Experimental Parasitol (1989) 68:176; Petralanda, I., et
al., ~Ql Biochem Parasitol (1986) 19:51). The protease
activity includes collagenase in the case of Bruqia
malayi, O. cervicalis and O. cervipedis. A collagenase
and a leucine aminopeptidase have been found in the
molting process of Haemonchus contortus by Rogers, W.P.,
W093/10225 ~ 1~ 3 4 ~ O PCT/US92/09702
-4-
J Parasitol (1982) 12:495, and Gamble, H.R., et al., Mol
Biochem PaxasitQl (1989) 33:49.
With respect to D. immii~is, protease activity
has been detected i~ soluble extracts of D. immitis
adults by Maki, J., et al., J Helminthol ~1986) 60:31-37,
and in extracts of microfilariae by Tomashiro, W.K., et
al., J Parasitol ~1987) 73:149-154. However, the soluble
extracts of D. immitis L3 and L4 and the excretory-
secretory ~E-S) products of these larval stages have not
previously been studied.
It is also known that collagens form the major
structural components of nematode cuticle by virtue of
studies conducted on Caenorhabditis eleqans, B. malayi,
and B. ~ahangi.
1~
Disclosure of the Invention
The invention i8 directed to prevention and
treatment of filarial nematode infection in animal hosts
and to purified and isolated forms of the proteases
~0 associated with the ~3 and L4 larval stages of the
parasites that cause these infections. One of these
nematodes is Dirofilaria immitis, which causes heartworm
; ~ in dogs. Other diseases of importance are caused by
nematodes such as tho~e listed above. The invention
provides an approach to the eradication of co~ditions
caused in animals by filarial nematodes, and provides
materials useful in these and in in vitro contexts.
Accordingly, i~ one aspect, the invention is
directed to a method to protect animal subjects,
including humans, against-filarial nematode infection,
which method comprises administering to the 8ub; ect an
effective amount of a metalloprotease and/or cysteine
protease characteristic of transition from the L3-L4
stage of the relevant filarial nematode effective to
immunologically protect the subject against infection.
WO93/10225 ~ d 3 ~ ~ ~ PCT/US92/09702
The characteristlc metalloprotease(s) may be found in the
L3 or L4 excretory-secretory material or in L3 or L4
lysates. The cysteine protease is found in L3 and L4
lysates.
In another aspect, the invention is directed to
the treatment of nematode filarial infection in animal
subjects, including humans, which method comprises
administering to that subject an effective amount of a
metalloprotease inhibitor and/or cysteine protease
inhibitor.
In other aspects, the invention is directed to
antibodies immunospecific for filarial L3 or L4
excretory-secretory products or L3 or ~4 lysate
metalloprotease(s) or to B3~or L4 lysa~e cysteine
protease(s) and to pharmaceutical compositions and
vaccines containing them.
In still another aspect, the invention is
directed to the L3/L4-associated metalloproteases and
cysteine proteases of filarial parasites in isolated and
purified form. These purified proteases are additionally
u~eful to assay for the presence or absence of a~ti~odies
i~ the diagnosis of affected individuals and to regulate
the growth of cell cultures in ~itro, as well as in other
therapeutic applications.
Brief D~ri ~ion of the Drawings
Figure 1 shows the elution pattern of protease
activity from L3/L4 E-S.
Figure~2 shows the elution pattern of protease
acti~ity from L4 lysate.
WO93/1022~ 212 3 ~ 2 0 -6- PCT/US92/09702
Modes of Carrying Out the Invention
As used herein, "metalloprotease" of ~3 and ~4
excretory-secretory preparation (L3 and L4 E-S) or of L3
or L4 lysates refers to metalloprotease enzymes
characteristic of the excretory-secretory products
obtained during the molting of the L3 larval stage into
L4 for filarial infective nematodes; or of whole worm
lysates of the L3 or L4 larval stage. At least one
"cysteine protease" is also found in ~3 and L4 lysates.
While the E-S and lysate preparations from D. immitis are
exemplified below, similar E-S or lysate preparations can
be obtained from various other filarial parasites such as
those set forth in the Background section ~bove, and
including, specificall~y, for example, B. malayi,
W. bancrofti, O. volvulus, Dipetalonema per~tans,
D streptocerca, Mansonella ozzardi, and Loa loa.
Preparation of~ Larval Cultures
The parasites can be cultured ln vitro under
suitable conditions~to provide a source for the E-S
preparation or for the L3 or L4 lysates. For example, D.
immitis can be cultured as described by Abraham, D., et
al., J Parasitol (1987) 73:377-383. Briefly, the
mos~uito Aedes :eqY~ti Liverpool (black-eyed strain) are
25~;~ infected with D. immitis by feeding on microfilaremic
blood obtaine~d f~rom a~single experimentally infected dog.
Fifteen days after feeding, the mosquitos are
anestheeized, surface sterilized and placed on screens in
funnels filled~with a l:l mixture of NCTC-135 and
Iscove' 8 modifisd~Dulbecco medium (Sigma) containing
2.5 ~g/ml amphotericin-B; lO0 ~g/ml gentamicin; 50 ~g/ml
sulfadiazone; and lO ~g/ml trimethoprim. The larvae are
collected from~funnels 90 minutes postincubation.
The cultures are maintained at a concentration
of ten L3 organisms per ml of medium in 5% CO2 and
.
2 ~ '1 2 0
WO93/10225 PCT/US92/09702
saturated humidity. The larvae (L3~ are cultured at 37
in the foregoing medium, supplemented with 20~ fetal calf
serum for l-~ days.
Alternatively, and preferably, lO days after
feeding, the mosquitos are anesthetized and the worms are
recovered by dissecting the heads and allowing the worms
to emexge into medium with 20~ Seru-max ~Sigma) to induce
molting. After 48 hr, the worms are recovered, washed 5
times in medium which does not contain Seru-max, and
recultured therein.
: :
Preparation of L3 and L4 E-S
L3 ES is collected between 4~ and 96 hours of
culture on Seru-max free medium. L4 ES is collected
between 96 and 144 hours in indentical culture
conditions. Medium containing ES is collected and
filtered through a 0.45 ~m filter. The ES is
concentrated and the buffer is exchanged into pH 7.2 PBS
using ultrafiltration and lO kd exclusion limit to obtain
the fraction of ~lO kd MW.
Pre~aration of L3 and L4 Lysates
Larval~soluble extracts are prepared from L3
collected on day 2, ~ust after the wash but prior to the
lt, and L4 are~collected on day 6 in serum-free
culture. Pellets of lO,000 worms in P~S are disrupted by
ten lO-sec high frequency pulses using a tissue
sonicator. Sonicated worms are centrifuged for 5 min at
: .
12,000 x g, and the supernatant collected~
Determination of E-S and Lysate Components
Protein concentration for both E-S and whole
worm soluble extracts may be estimated using a Micro BCA
kit (Pierce Chemical Co., Rockford, IL). All samples are
maintained at -20 C prior to further analysis.
W093/10225 PCT/US92/09702
2123 ~120 -8-
By "metalloprotease" of the L3 and L4 E-S
preparation or of L3 or L4 lysates is meant a protease
enzyme which is found in the excretory-secretory product
of third or fourth stage lar~ae or in L3 or L4 lysates of
a filarial nematode parasite as ascertained by acti~ity
against the synthetic substrate h-phenylalanine-AMC (h-F-
AMC, defined below) and which is inhibited by metallo-
protease inhibitors such as 1,10-phenan~hroline and EDTA.
Metalloprotea~e activity has been reported in E-S
products of third stage larvae of certain species,
including B. malayi, O. cervicalis, and O. cervipedis as
set forth above. The activity is also present in L3 and
L4 lysates.
The invention also relates to "cysteine
protease(s)" from L3 or L4 lysates, which lysates may be
prepared as described above. The cysteine proteases of
the invention are characterized by ability to hydrolyze
Z-valine-leucine-arginine-AMC tZ-VLR-AMC, defined below)
and this acti~ity is inhibited by E64. Again, D. immitis
is used for illustration below, but other filarial
nematodes may be used.
Both of these enzymes may be obtained in
purified and isolated form using chromatographic me~hods
with use of the appropriate substrate as~ay to monitor
elution fractions as further described below.
~ ecause of the practical difficulties in
obtaining sufficient quantities of the metalloprotease of
L3 and L4 E-S preparations or the metalloprotease or
cysteine protease from ~3 or L4 lysates to provide
material for vaccines, alternati~e methods of production
are preferred when large quantities are desired.
Specifically, for the full-length metalloprotease or
cysteine protease, recombinant production is the most
practical approach; for immunogenic subunits which are
capable of eliciting antibodies that neutralize the
metalloprotease or cysteine protease activity, ordinary
solid phase peptide synthesis may be preferred. However,
eYen in ~his instance, it may be desirable to utilize
recombinant production to obtain tandem repeats of the
immunogenic subunit. Production of tandem repeats may
enhance the immunogenicity of the material. In addition,
the subunit vaccines may be recombinantly produced as
fusion proteins to an immunogenicity-conferring sequence.
Recombinant Production
The recombinant sequences necessary for
production of the relevant metalloprotease or cysteine
protease are obtained in a process analogous to that
described by Sakanari, J.A., et al., Proc Natl_Acad Sci
(1989) 86:4863. In this process, the gene encoding the
metalloprotease or cysteine protease is isolated from
cDNA prepared from total mRNA o~ the L3 or L4 stage of
the parasite using oligonucleotide primers and the
polymerase chain reaction (PCR) and suitabie probes.
D. immitis genomic or cDNA is used as a ~ource
;for protease-encoding genes. For isolation of the
metàlloprotease gene, primers are designed based on
consensus sequences in the bacterial metalloprotease
thermolysin, and~members of the human metalloprotease
family which include stromelysin, stromelysin II, and
Pump-I. These highly ho logous gene~ are all
metalloproteases, and the cDNAs containing these
equences ha~e been disclosed (Muller, D., et al.,
Biochem J ~I988) 253:187-192 and Quantin, B., et al.,
ioch~mist~y (1989) 28:5327-5334). Primers can be
designed based on the conserYed regions, including the
active site. PCR amplification is conducted a~ described
by Sakanari et al. ~supra), and reaction products are
loaded on agarose/NuSieve (FMC). An additional probe
designed based on the above-mentioned published sequences
WO93/10225 2 1 ~ ~ 1 2 0 - lo - PCT/US92/09702
is used to identify the relevant amplified gene products
on Southern blots. The fragments are cut out of the gel,
extracted with ~glass milk" (Geneclean) and liga~ed into
Bluescript ~Stratagene) to obtain sequences of both
coding and anticoding strands, using the dideoxy method
of Sanger with Sequenase (USB) and the SK and KS primers
for sequencing in both directions. The gene fragments
obtained are then u~ed as probes to screen a cDNA
~ibrary. They are labeled with 32p using standard random
priming methods.
For preparation of analogous probes for the
cysteine protease genes, primers are de~igned based on
the sequences disclosed in Eakin, A.E. et al., ~1
Biochem Parasitol (1~90) 39:1-8. Otherwise, the
retrie~al of probes from genomic DNA can be conducted as
abo~e.
The cDNA library is construc~ed from messenger
RNA i~olated from third stage larvae which have been in
culture for 4~-72 hours. The mRNA is isolated by the
sing}e step acid guanidinium thiocyanate/phenol/
chloroform extraction method of Chomczynski, P. and
Sacchi, N., Anal Biochem (1987) 162:156-159. The RNA is
passed over an oligo-dT cellulose col~mn and the poly-A
RNA is eluted using ~andard procedures. cDNA is
prepared from the mRNA using sta~dard procedures such as
tho~e of Gubler, U. and Hoffman, B.J., Gene ~1983)
~:263. The cDNA is treated by methylation of internal
EcoRI sites, a~d phosphorylated EcoRI linker~ are added
to the ends of the cDNA and trea~ed again with
pho~phatase. The treated cDNA contain linkers diges~ed
with EcoRI to generate cohesive cloning ends for
in~ertion in~o A-gtll arms (Stratagene, San Diego, CA)
and packaged using Gigapack (Stratagene). Standard
methods are used to titer and plate the library for
screening.
- ~1.?3'l2~
WO93/10225 -11- PCT/US92/09702
The library can be screened either using the
probes obtained as described above, heterologous probes,
or the expression products can be screened using
antibodies prepared against the proteases obtained from
the E-S product or lysates. Selected clones are plaque
purified, and the isolated coding sequences are used to
produce the recombinant protease.
The cloned DNA can be used directly in
expres~ion vectors, or DNA can be synthesiæed using
standard 901 id phase techniques to obtain any embodiment
o~ the coding sequence to supply all or a portion of the
gene.
For example, a DNA coding sequence ~or the
protease can be prepared synthetically from overlapping
oligonucleotides who~e -Qequence contains coduns for the
amino acid sequence encoded in the native gene. Such
oligonucleotides are prepared by standard methods and
as~embled into a cGmplete or partial coding sequence.
See, e.g., Edge, Nature (1981) 2~2:756; Nambair et al.,
Science (1984) 223:1299; Jay et al., J Biol Chem (1984)
259:6311.
Thus, a DNA molecule containing the coding
~e~uence for the filarial nematode metalloprotease or
cysteine protease can be cloned in any suitable vector
and thereby maintainéd in a compo~ition substantially
free of vectors that do not contain the coding ~equence
for the protease (e.g., other library clones). Numerous
clo~ing vectors are known to those of skill in the art,
and the Yelection of an appropriate cloning vector is a
matter of choice. Examples of recombinant DNA vec~ors
for clo~ing and the host cells which they transform
include bacteriophage ~ (E. coli), pBR322 (E. coli),
pA~YC177 (E. coli), pKT230 (gram-negative bacteria),
pBG1106 (gram-negative bacteria), pLAPR1 (gram-negative
bacteria), pME290 (non-E. coli gram-negative bacteria),
WO93/10225 PCT/US92/09702
2~23~12~ -12-
pHVl4 (E. coli and Bacillus subtilis), pBD9 (Bacillus),
pIJ6l (Streptomyces), pUC6 (Streptomyces), actinophage
~C31 (Streptomyces), YIp5 (yeast), YCpl9 (yeast), and
bovine papilloma virus (mdmmalian cells).
For expression, the coding sequence of the
protea~e gene is placed under the control of a promoter,
ribosome binding site (for bacterial expression) and,
optionally, an operator (collectively referred to herein
a~ ~Icontrol~l sequences) so that the protease-encoding
sequence is transcribed into RNA in the host cell
transformed by the vector. The coding sequence may or
may not contain a signal peptide or leader sequence. In
bacteria, the protease is preferably produced by the
expression of a coding sequence which does not conSain
any ~ative signal peptide, or by expression of a coding
sequence containing the leader sequence in a eucaryotic
system when post-translational processing removes thP
leader sequence. The protease can also be expressed in
the form of a fu~ion protein, wherein a heterologous
amino acid sequence is expressed at the N- or C-terminus.
See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437.
The recombinant vector is constructed so ~hat
the protease-encoding sequence is located in ~he vec~or
with the appropriate control sequences, the positioning
and orientation of the coding sequence with respect to
the control sequences being such that the coding ~equence
i9 transcribed under the control of the control sequences
(i.e., by RNA polymera~e which attaches to the DNA
molecule at the control sequences). The control
~equences may be ligated to the coding sequence prior to
in~ertion into a vector, such as the cloning ve~tors
described above. Alternatively, the coding ~equence can
be cloned directly into an expreYsion ~ector which
already contains the control sequence and an appropriate
restriction site downstream from control sequences. For
WO93/10225 ~12 3 4 2 ~ PCT/US92/09702
-13-
expression of the protease-encoding sequence in other
than nematodes, the control sequences will be
heterologous to the coding sequence. If the host cell is
a procaryote, it is also necessary that the coding
~e~uence be free of introns; e.g., cDNA. If the selected
host cell is a nematode cell, the control sequences can
be heterologous or homologous to the protease-encoding
sequence, and the coding sequence can be genomic DNA
containing introns or cDNA. Either genomic or cDNA
coding sequences may be also expressed in yeast.
A number of procaryotic expression vectors are
known in the art. See, e.g., U.S. Patent No~. 4,440,B59;
4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437;
4,418,149; 4,411,994; 4,366,246; 4,342,832. Preferred
expression vectors, however, are those for use in
eucaryotic systems. Yeast expre~sion vectors are ~nown
in the art. See, e.g., U.S. Patent Nos. 4,446,235;
4,443,539; 4,430,428. See also European Patent
Specifications 103,409; 100,561; 96,491. The recombinant
protease can be produced by growing host cells
tra~sformed by the expression vector described above
under conditions whereby the protease is produced. Human
collagenase cDNA has been cloned and expressed in active
form in eucaryotic cells (Muller, D., et al., Biochem J
(I988) 2$3:187-192). The protease is then isolated from
the host cells and purified. If the expre~sion system
- secretes the protease into growth media, the desired
protein can ~e purified directly from cell-free media.
If the protease is not secreted, it is isolated from cell
lysates. The selection of the appropriate growth
conditions and recovery methods are within the skill of
the art; purifications similar to those exemplified below
can be used.
WO93/1~22S PCT/US92/097~2
2 1 2 3 Ll 2 ~ - 14-
Antibody Production
Either nati~e or recombinant proteases of the
invention can be used to produce antibodies, both
polyclonal and monoclonal. If polyclonal antibodies are
desired, the purified protease is used to immunize a
selected mammal (e.g., mouse, rabbit, goat, horse, etc.)
and serum from the immunized animal later collected and
treated according to known procedures. Compositions
containing polyclonal antibodies to a variety of antigens
in addition to the relevant protease can be made
substantially free of anti~odies which are not protease
antibodies by passing the composition through a column to
which the desired protease has been bound. After
washing, polyclonal antibodi~es are eluted from the
column. Monoclonal antibodies can also be readily
produced by one skilled in the art. The general
methodology for making monoclonal antibodies by
hybridomas is well known. Immortal, antibody-producing
cell lines can also be created by techniques other than
fusion, ~uch as dire~t transformation of B lymphocytes
with oncogenic DNA, or transfection with Epstein-Barr
virus. See, e.g., Schreier, M., et al., HYBRIDOMA
TRCHNIQUES (1980); Hammerling et al., MONOCLONAL
ANTIBODI~S AND T-CE~L HYBRIDOMAS (l98l~; Kennett et al.,
MONOCLONAL ANTIBODIES (1980).
-By employing the metalloprotease or cysteine
protease (native or synthetic) as an antigen in the
immNnization of the source of the B-cells immortalized
for the production of monoclonal antibodie~, a panel of
monoclonal antibodies recognizing epitopes at different
sites on the protease can be obtained. Antibodies which
recognize an epitope in the active site binding region of
the protease can be readily identified in competition
assays between antibodies and enzyme substrate.
Artificial substrates such as Z-VLR-AMC ~cysteine
.
W093/10225 2 1 2 3 1 2 0 PCT/US92/09702
-15-
protease) or h-F-AMC (metalloprotease) can also be u~ed.
Such an~ibodies have therapeutic potential if they are
able to block the binding of protease to its substrate n
vivo. Antibodieg which recognize a site on the protea~e
S are also useful, for example, in the purification of the
desired protease protein from cell lysates or
~ermentation media, and in its characterization. In
general, as is known in the art, the protease antibody is
fixed (immobilized) to a solid support, such as a column
or latex beads, contacted with a solution containing the
protease, and separated from the solution. The protea~e,
bound to the immobilized antibodies, is then eluted.
Isolation and Purification of the L3 and L4 Proteases
~s stated above, the cysteine protease
characteristic of the L3 and L4 lysates and the
metalloprotea~e characteristic of these lysates, a~ well
as the L3/L4 E-S, can be obtained in isolated and
purified form either using the appropria~e larval stage
of the desired parasitic nematode as starting material,
using recombinant production in cell culture and
isola~ing the protease resulting from the cysteine
protease or metalloprotease gene expression, or by
~ynthesiziny 8ubunit8 of these proteins using standard
peptide syntheQis techniques. The na~ure of the
purification method will depend on the origin of the
protease or peptide.
When isolated from natiYe sources, the lyqate
or E-S materiaI i9 ~ubjected to chromatographic
technigues, typically chromatography using affinity
chromatography (e.g., affinity chromatography using
antibodies prepared with respect to the protease as
affinity ligands), ion-exchange chromatography, sizing
columns, reverse-phase colu~ns, and the like.
Optimization of the purification procedure is within the
WO93/10225 2 1 2 3 ~ 2 0 - 16- PCT/US92/09702
skill of the art, as the fractions eluted from the
columns can be assayed using activity determination with
a fluorometric substra~e characteristic of the
metalloprotease or cysteine protease. For the
metalloprotease of ~ immitis, h-F-AMC is a convenient
~ubstrate; for the cysteine protease of thiY worm,
Z-V~R-AMC is appropriately used. The specificity and
nature of the protease can be verified by supplementing
the assay with various inhibitors known to characterize
metalloproteases or cysteine proteases. Modified forms
of these substrates may be appropriate for the
~etalloproteases or cysteine proteases of other species
of filarial nematodes; the appropriate substrate can be
ascertained by the conduct of preliminary assays on the
crude extracts, as exemplified herein ~or the D. immitis
species .
If the protease is produced recombinantly,
similar techniques can~be used, although the starting
material generally contains the protease in a more highly
concentrated form. ~Further modification of the
purification procedure is appropriate for isolation of
the peptides prepared by solid-phase synthesis, since the
nature of the contaminants is different. Generally,
dialysis or other~size-separation methods are
appropriate.
The purified and isolated ~orms of the cy3teine
and metalloproteases of the various filarial n~matode
species can be used in the production of antibodies
(which antibodies, in turn, are useful in immunoassays
and separation techniques), as reagents in ~mmunoas~ay
procedures for the presence or absence of antibodies, and
; in the regulation of cell culture in vitEo by controlling
extracellular matrix formation or status.
::
'
WO93/10225 ~ 3 '~ 2 0 PCT/US92/09702
U~e of Purified and Isolated Proteases in Diaqno~is
The purified and isolated proteases are u~eful
in diagnostic immunoassays for the presence or absence of
antibodies with respect to filarial nematode species.
These assays can be used to assess the disease state of a
host organism or to assay titers in immunization
protocols. The assays are conducted in standard
immunological format, including RIA, ELISA, and
fluorescence-labeled assays. The assays can be conducted
in either a direct or a competitive format and rely on
~eparations by virtue of binding to solid support or by
virtue of precipitation of immunological complexes. A
large number of protocols suitable for the conduct of
immunoa~says i9 well known in the art.
U~e of Purified and Isolated Proteases in Vacçines
;~ The ?roteases of the invention are useful as
vaccines in immunizin~ host organisms to protect them
against i~fection by the corresponding filarial nematode.
The proteases are administered in standard pharmaceutical
formulations ystemically, and typically by injection.
Injection may be~intravenous, intramuscular, peritoneal,
or other parenteral. Suitable vehicles for in~ection
include physiological saline, Hank's solution, Ringer's
solution and the like, with or without the pre3ence of
adjuvants, according to the immunization protocol.
Generally, the vaccine is administered at a dosage level
sufficient to raise~antibody titers to pro~ide effective
cavenging of the proteases required for molting from the
~3 ~o the ~4 stage in the filarial infecti~e agent.
A
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WO93/10225 2 1 2 3 ~ 2 0 -18- PCT/US92/09702
Treatment of ~nfection with Inhibitors
As the proteases of the invention are needed
for the progression of the parasitic nematode life cycle,
administration of inhibitors of these enzymes to infected
hosts in suita~le do ages inhibits or arrests the course
of the infection. The inhibitors are formulated in
~uitable pharmaceutical compositions such as those
described in Remin~ton's Pharmaceutical Scien~es, latest
edition, Mack Publishing Co., Easton, PA. Administration
10 i9 preferably by oral formulation, although injection or
transdermal or transmucosal routes can also be used.
The following examples are intended to
illustrate, ~ut not to limit the invention.
Example 1
Demon~tration of Protease_Rctivity in D. Immitis
D. immitis were cultured at a concentration of
lO0 larvae per ml in a 1:1 mixture of NCTC-135 and
IYcove' 8 modified Dulbecco medium (Sigma) containing
antibiotics (NI) on a model of extracellular matrix (ECM)
secreted by rat vascular smooth muscle cells and labelled
with tritiated proline. Every 8 hrs a 50 ~l sample was
collected and the amount of tritium released from the
matrix was counted on a scintillation counter. The
counts per minute of tritium released from the ECM for
the L3 stage increased 810wly from 1 x 104 cpm after 8
hours to about 2 x 104 cpm after 56 hours, when L3
molting occurs.~ A large incremental release of tritium
(indicating degradation of the matrix) occurs at the time
of L3 molting; cpm increase to about 6 x 104 cpm after 64
hrs and to over 8 x 104 cpm after 72 hrs. The breakdown
of matrix mediated by L4 tracked that by ~3 until the 56
hour L3 molt event; cpm for L4 continued to increase only
slowly after this (to ~4 x 104 cpm after 72 hrs). In
~093/10225 2 ~ 2 3 d 2 J~ PCT/US92/09702
total, a~ter 72 hours the L3 culture d~graded 20% of the
total ECM, and the L4 culture degraded 13%.
ThP components of the ECM which were degraded
were evaluated by sequential enzyme digests of the
remaining ECM as described previously (McKerrow, J.H., et
al., Lab Invest (1933) 49:195-200.
The results are shown in Table 1. Collagen is
shown to be the major component of the ECM degraded by
both L3 and ~4 lysates. However, L3 lysates degrade
nearly twice as much collagen as lysate from ~4
Table 1
Percent Deqradation of ECM Constituent Proteins
(100 larvae lysate/ml)
~3 4
; Glycoproteins 22 20
Elastin 10 8
Collagen 61 38
In these experiments, controls for nonparasite-
derived degradation of ECM constituted either NI alone,
mosquito media, or C. ele~ans. Mosquito media were
prepared from noninfected mosquito heads processed as if
they contained worms. C. elegans adults and larvae were
recovered from NGM agarose plates seeded with E. cQli
strain OP50, placed in M9 media at the same concentration
as the ~ immitis larvae and inc~bated at 26C. Mosquito
media were u~ed as a control to aScure that mosquito-
; 30 derived proteases were not responsible for any of the
degradation observed. C. elegans, a free-living
nematode, was used as a comparison to tissue-invasive
nematodes and as a control to insure motility alone did
not cau~e release of label.
W093/10225 2 l 2. ~ 4 2 ~ PCT/US92/0975~
-20-
Lysates from L3 and L4 were prepared by
~onication of the larvae in PBS on ice using 10 x 10 sec
high frequency pulses. Lysates containing 10 ~g protein
per reaction were tested against artificial substrates
con8isting of amino acids linked to a fluorogenic
; compound, 7-amido-4-methylcoumarin (AMC) (Bachem). Some
substrates were protected against exopeptidase acti~ity
by a benzyloxycarbonyl group, abbre~iated Z; the
substrates that are not protected are indicated by a
preceding "h". These substrates are Z-Val-Leu-Arg-AMC,
h-Phe-AMC, Z-Phe-Arg-AMC, and Z-Arg-Arg-AMC (abbreviated
; Z-VLR-AMC, h-F-AMC, Z-FR-AMC, and Z-RR-AMC respectively).
The lysate was incubated with each substrate for 3 hrs,
and the amount of AMC hydrolyzed was measured
fluorimetrically. Cleavage of AMC was measured using an
LS-2 ~pectrofluorometer (Perkin Elmer) with 380 nm
excitation wavelength and e~ission detection at 460 nm.
Initial substrate screening reactions consisted of 10 ~1
~ubstrate (at 5 mM in DMS0), 980 ~1 PBS, pH 7.2, and
10 ~1 combined L3 and L4 sol~ble extracts. A number of
additional sub~trates, found to be uncleaved, were also
tested: Z-GPLGP-AMC, Z-GPR-AMC, Z-ARR-AMC, Z-AR-AMC, Z-
R-AMC and h-L-AMC. However,;further work was conducted
with the four substrates listed above. Two mM dithio-
threitol (DTT)~was found to enhance clea~age of Z-VLR-AMC
wofold, but to~inhibit h-F-AMC hydrolysis as shown in
Tàble 2. Thus, Z-VLR~AMC i8 shown to be a substrate for
this cysteine protease, h-F-AMC is a substrate for the
metalloprotease.
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WO93/1022~ 2 ~ 2 ~ ~ 2 ~ PCT~US9~/09702
Table 2
Effect of 2 mM DTT on Hydrolysis of
R-AMC and h-F-AMC by L3 Soluble Extract
Substr~e nMoles AMC released/hr
+DTT -DTT
h-Phe-AMC 99.0 (16.7) 326.4 (25.0)
z-Val-Leu-Arg-AMC 54.6 (1.7) 32.6 (2.6)
Data represent the means of duplicate samples with ranges
indicated in parentheses.
.
Thereafter, in the determinations, the P~S
contained 2 mM DTT except when h-F-AMC was u~ed.
Further, reaction mixtures consisted of 10 ~1 5
~ ~ mM substrate, 10 ~1 larval soluble extract or E-S at
;~ protein concentration of 1 ~g/ml and 980 ~1 of PBS, pH
7.2. After i~cubation of the lysate or E-S with
3ubstrate at 37C~for a specified length of time, the
hydrolyzed AMC was measured on a Perkin Elmer LS-2 filter
fluorometer with excitation and emission wavelength~ as
set forth abo~e. For h-F-AMC, the L3 lysate relea~es
about~20 ~mol of AMC after the 3 hr incubation while the
~4 lysate releaYes slightly less than 10 ~mol. Z-FR-AMC
a~d Z-RR-AMC are not effective substrates ~or either
lysate; approximately 5 ~mol of AMC are released from Z-
VLR-AMC by either extract.
The excretory-secretory materials were also
tested for activity on these substrates. Using 2 ~g of
protein per reaction, the L3 E-S composition released
about 9 ~mol AMC from h-F-AMC per reaction mixture after
3 hr whereas the L4 E-S composition released only about 2
W O 93/10225 2 1 2 ~ 4 2 0 -22- PC~r/VS92/09702
~mol. No AMC was released from the Z-VLR-AMC, Z-FR-AMC
or Z-RR-AMC substrates.
The effect of inhibitors was also tested with
respect to the synthetic substrates Z-VLR-AMC and h-F-AMC
using the lysates of L3 and L4 prepared as aboYe.
h-F-AMC wa~ shown to be a substrate for this
metalloprotease; Z-VLR-AMC was shown to be a substrate
for this cysteine protease (DTT enhances cysteine
protease acti~ity; oxidizing conditions inhibit it) (see
Table 2). Ten ~l (10 ~g total protein~ of L3 or L4
lysate was mixed with 10 ~l 50 ~M synthetic peptide
substrate, 960 ~l PBS buffer, pH 7.2 containing 2 mM DTT
when Z-VLR-AMC was used and without DTT when h-F-AMC was
u~ed. Twenty ~l of the test~inhibitors were added to the
reaction mixtures, the final concentrations of the
inhibitors were as follows: PMSF, 2 mM; l,10-phenanthro-
line, 10 mM; NEM, 2 mM; E-64, 10 mM; Bestatin, 1 mM;
Cystatin, 4 ~M; and EDTA, 2 mM. Inhibition was
calculated as the percent activity remaining as compared
to control in absence of the inhibitor. The results are
; shown in Table 3 ~L3) and Table 4 (L4~.
Table 3
L3 hYsate % Control Acti~ity Remaininq
~ Substrate
Z-VLR-AMC h-F-AMC
PMSF 63 11
NEM : 54 22
E64 12 100
1,10-Phe . 31 8
Bestatin 62 100
Cystatin 60 100
EDTA 21 5
WO93/1022~2 1 2 3 ~1 2 0 PCT/US92/09702
-23-
Table 4
L4 h~sate ~ Control Activity Remaininq
Substrate
Z-VLR-AMC h-F-AMC
S PMSF 92 20
NEM 20 23
E64 15 7~
1,10-Phe 24 5
Bestatin 80 88
Cystatin 60 97
EDTA 18 4
E64, a potent cysteine protease inhibitor, had
e~sentially no effect on th~ metalloprotease substrate
h-F-AMC; however, E64 was the most effective inhibitor
for the cy~teine protease substrate, Z-V~R-AMC.
The activity of L4 lysates with respect to the
various fluorogenic synthetic substrates was also te~ted
in the presence and ab~ence of DTT. DTT seemed to
enhance the activity with respect to Z-VLR-AMC, Z-FR-AMC
;:
and Z-RR-AMC. DTT i~ known to enhance the activity of
cysteine proteases and to inhibit metalloprotea~es.
The effect of the same inhibitor~ was tested
essentially as de~cribed above with respect to the
hydroly~is of h-F-AMC by L3 E-S. The reactions contain
10 ~1 ~3 ~-S, i.e., 10 ~g total protein, 10 ~1 of the
h-F-AMC to gi~e 50 ~M final concen~ration, 960 ~1 PBS pH
7 ~ 2 and 20 ~ hibitor to give final concentrations as
for the ly~ate~ above. The inhibition pattern, as shown
in Table 5, is imilar to that shown by the lysates.
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~ 35
W093i10225 2 ~ 2 ~ 4 2 n -24- PCT/US92/09702
Table 5
E-S from L3, ~ of Con~rol ~cti~ity Remaininq
Substrate
h-F-AMC
PMSF 22
NEM 26
E64 79
l,lO-Phe 5
Bestatin 80
ln Cystatin 98
EDTA 5
Taken together, these data show that the
ly ates and ~-S preparations~contain metalloprotease, but
only the ly~ates contain ~ignificant amounts of cysteine
protea~e activity.
Exam~le 2
Pre~aration of_Pr~teases from D. Immitis
The L3 or L4 ly~ates or the h3/h4 E-S prepared
as described above were ~ubjected to size exclusion
chromatography.
The ~3/L4 E-S of 8,000 larvae, collected from
~:: 48 to 144 hours, was concentrated to 7S ~l in 0.05 M
Tris/HCl, pH 6.8, O.lS M NaCl, and injec~ed for size
exclusio~ chromatography into a TSK 3,000 SW 7.5 x 300 mm
column with a 7.5 x 75 mm guard column (Beckman,
: : Fullerton, C~) attached to Beckman Model 338 HP~C. The
mobile pha~e used the ~ame buffer, the flow rate was 0.5
ml/min a~d ~he detector was set at 220 nm. O~e mi~ute
fractio~ were collected starting at 12 minutes. The
: column was calibrated using gel fil~ration molecular
weight markers ~NW-GF-200) (Sigma).
:~ The L3/L4 E-S which had been metabolically
labeled with S35 methionine and cysteine was collected
.
21~3'~2~
~WO93/10225 -25- PCT/US92/09702
and chromatographed under the conditions described above
and the fractions were subjected to reducing SDS-PAGE
using standard techniques. Figure 1 shows the
chromatogram obtained when h-F-AMC was used as a
substrate to assay activity of the fractions--20 ~l of
each fraction was incubated with S mM h-F-AMC in 970 ml
PBS, pH 7.2, for 1 hour. Peak enzyme acti~ity was in
fraction 10 which correspQnded to a molecular weight of
approximately 49-58 kd. SDS-PAGE analysis of fraction 10
gave three prominent bands at 5~, 30 and 22 kd and three
minor bands at 28, 26 and 19 kd under denaturing and
.
reducing conditions.
Similar separations were run using lysates
prepared from 6,000 L4 worms collected after 144 hours.
The fractions were assayed using both Z-VLR-AMC and
h-F-AMC as sub~trates. The results are ~hown in Figure
2. The h-F-AMC~acti~ity (metalloprotease) eluted at a
,~
position corresponding to 49-54 kd; the Z-VLR-AMC
acti~ity eluted at a position corresponding to 31-34 kd.
2~
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