U.S. patent application number 15/498066 was filed with the patent office on 2017-10-26 for genetically modified babesia parasites expressing protective tick antigens and uses thereof.
The applicant listed for this patent is The United States of America as represented by the Secretary of Agriculture, The United States of America as represented by the Secretary of Agriculture, Washington State University. Invention is credited to Donald P. Knowles, Jr., Terry McElwain, Carlos E. Suarez.
Application Number | 20170304416 15/498066 |
Document ID | / |
Family ID | 40136741 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170304416 |
Kind Code |
A1 |
Suarez; Carlos E. ; et
al. |
October 26, 2017 |
GENETICALLY MODIFIED BABESIA PARASITES EXPRESSING PROTECTIVE TICK
ANTIGENS AND USES THEREOF
Abstract
The present invention relates to methods for stable transfection
of Babesia parasites, and for vaccines conferring immunity against
parasitic arthropods.
Inventors: |
Suarez; Carlos E.; (Pullman,
WA) ; Knowles, Jr.; Donald P.; (Pullman, WA) ;
McElwain; Terry; (Pullman, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America as represented by the Secretary of
Agriculture
Washington State University |
Washington
Pullman |
DC
WA |
US
US |
|
|
Family ID: |
40136741 |
Appl. No.: |
15/498066 |
Filed: |
April 26, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14995870 |
Jan 14, 2016 |
9707283 |
|
|
15498066 |
|
|
|
|
12114988 |
May 5, 2008 |
9265818 |
|
|
14995870 |
|
|
|
|
60927800 |
May 4, 2007 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/522 20130101;
C12N 15/79 20130101; A61K 39/0003 20130101; C07K 14/43527 20130101;
A61K 39/018 20130101; A61P 33/02 20180101; A61K 2039/523
20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/018 20060101 A61K039/018 |
Claims
1-58. (canceled)
59. A method for generating an immune response in a mammal against
a heterologous protein, the method comprising administering to said
mammal a genetically modified Babesia spp. that produces said
heterologous protein, wherein said genetically modified Babesia
spp. comprises a stably transfected polynucleotide, wherein said
stably transfected polynucleotide comprises a promoter operably
linked to a heterologous nucleic acid, wherein said heterologous
nucleic acid encodes said heterologous protein, wherein said
promoter is transcriptionally active in said Babesia spp., and
wherein said mammal generates an immune response to said
heterologous protein produced by said genetically modified Babesia
spp.
60. The method of claim 59, wherein said genetically modified
Babesia spp. are Babesia spp. merozoiotes or Babesia spp. infected
red blood cells.
61. The method of claim 59, wherein said heterologous protein is a
tick protein.
62. The method of claim 61, wherein said tick is Boophilus spp. or
Ixodes spp.
63. The method of claim 59, wherein said stably transfected
polynucleotide further comprises a targeting sequence.
64. The method of claim 63, wherein said targeting sequence is part
of a gene that is not needed by said Babesia spp. for growth
in-vitro.
65. The method of claim 59, wherein said stably transfected
polynucleotide further comprises a selectable marker.
66. The method of claim 59, wherein said stably transfected
polynucleotide further comprises a targeting sequence and a
selectable marker.
Description
CROSS-REFERENCE TO PRIOR FILED APPLICATIONS
[0001] This application is a divisional patent application of U.S.
patent application Ser. No. 14/995,870 filed on Jan. 14, 2016
(allowed) which is a continuation patent application of U.S. patent
application Ser. No. 12/114,988 filed on May 5, 2008, now U.S. Pat.
No. 9,265,818, which claims benefit of U.S. Patent Application
60/927,800 filed May 4, 2007, all of which are incorporated herein
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to vaccines that are protective
against parasitic arthropods.
BACKGROUND OF THE INVENTION
[0003] Numerous species of arthropods are parasitic, and many play
a role in transmission of disease. Indeed, parasitic arthropods and
the diseases they transmit are a global problem. Ecto-parasitic
arthropods such as, e.g., ticks, mites, flies, fleas, midges, suck
blood from their hosts and in the process, can act as vectors for
protozoan, rickettsial and viral pathogens. Thus, the presence of
ecto-parasitic arthropods is frequently associated with
disease.
[0004] Among the ecto-parasitic arthropods, ticks are particularly
problematic and harmful. Indeed, ticks are second only to
mosquitoes as vectors of human disease, both infectious and toxic.
Hard ticks (Ixodidae) can transmit human diseases such as e.g.,
relapsing fever, Lyme disease, Rocky Mountain spotted fever,
tularemia, equine encephalitis, Colorado tick fever, and several
forms of ehrlichiosis. Additionally, they are responsible for
transmitting livestock and pet diseases, including babesiosis,
anaplasmosis and cytauxzoonosis.
[0005] Because of their ability to transmit diseases to humans and
animals, the medical and economic importance of ticks has long been
recognized. Economic losses associated with ticks are typically
manifest through their adverse effects on their livestock hosts.
See e.g., L'Hostis M, Seegers H. (2002) Vet Res. 33(5):599-611;
Peter, R. J., et al. (2005) Vet Parasitol. 132(3-4):205-215. In
addition to being disease vectors, blood sucking by large numbers
of ticks can cause a loss of blood in the host animal. This, in
turn, can result in a reduction in live weight, and may even result
in anemia. Still more, multiple tick bites can reduce the quality
of hides. Thus, ticks affect the product performance of
livestock.
[0006] Ticks and tick-borne diseases are important in all domestic
animals, but the development and production of innovative tick
control methods have been focused primarily on the economically
important tick-borne diseases of cattle. Indeed, the tick borne
protozoan Babesia parasites remain an important limitation for
development of cattle industries worldwide. Effective control of
Babesia and other tick borne diseases will certainly require
eradication of the tick vectors as well as vaccination against the
Babesia parasites.
[0007] Given the impact tick infestations can have on livestock, it
is not surprising that numerous methods for tick control have been
attempted see e.g., U.S. Pat. No. 6,103,758, U.S. Pat. No.
6,331,297, U.S. Pat. No. 6,100,501 and U.S. Pat. No. 5,587,311.
Unfortunately however, every method so far developed has
shortcomings that limit wide application of the method.
[0008] For example, chemical acaricides have traditionally been the
first line defense against ticks, and do show efficacy.
Unfortunately however, the use of chemical acaricides has numerous
drawbacks, including, but not limited to the development of
chemical resistant tick strains, the presence of residues in the
milk and meat, and harmful effects on the animals being treated,
human beings, and the environment (see e.g., Nolan J. (1990)
Parasitol. 32:145-153; George J. E., et al. (2004) Parasitology.
129(7):5353-5366; Wharton, R. H., and Roulston, W. J. (1970) Annu
Rev Entomol. 15(1):381-405; and U.S. Department of Agriculture.
Agriculture Handbook, No. 321. Washington, D. C: 1967. Safe Use of
Agricultural and Household Pesticides; p. 65.).
[0009] Because of the problems associated with use of chemical
acaricide products, alternative methods for tick control have also
been used and/or tested. For example, resistance to tick
infestation varies among individual animals and among different
breeds of cattle (see e.g., Latif, A. A., and Pegram, R. G. (1992)
Insect Sci & Appl. 13:505-513). Therefore, breeding of tick
resistant cattle has been attempted (see e.g., Wharton, R. H.
(1983) Wld Anim Rev, (FAO). 36:34-41). However, despite the
attractiveness of this approach to tick control, selective breeding
for tick resistance is difficult, unpredictable, and time
consuming. Indeed, each animal still develops its own level of
resistance in response to tick challenge and a wide range of
resistance occurs. Resistance can only be tested by exposing the
putatively resistant animals to ticks, and then resistance can only
be measured in terms of average number of ticks per animal. Thus,
development of new resistant breeds is a time consuming process
with limited usefulness.
[0010] Despite the difficulties associated with breeding resistant
livestock strains, the idea of tick resistant animals remains
attractive. Therefore, attempts have been made to achieve
resistance through vaccination. Indeed, a number of vaccines
against ticks and tick-borne diseases have been developed or are in
the course of being developed. Vaccines have utilized complex tick
extracts to stimulate an acquired immunity (see e.g., Willadsen,
P., and Kemp, D. H. (1988) Parasitol Today. 4(7):196-198). And,
isolated tick proteins such as Bm86, and Bm95 have been used for
the production of recombinant vaccines (see e.g., Willadsen, P., et
al. (1988) Int J Parasitol. 18(2):183-189; Rand, K. N., et al.
(1989) Proc Natl Acad Sci U S A. Dec;86(24):9657-61; Garcia-Garcia,
J. C., et al. (2000) Vaccine. 18(21):2275-2287; Willadsen P. (2004)
Parasitology 129 Suppl:S367-87 Review; and de la Fuente, J. et al.
(1999) Genet Anal. 15(3-5):143-8. Review).
[0011] Unfortunately however, widespread use of recombinant
vaccines is limited by a number of factors. First, vaccines,
recombinant or otherwise, must be produced in large fermentors.
Recombinant proteins and/or other antigens must be isolated,
typically requiring cumbersome methods, and the isolated antigens
may not be completely pure. Furthermore, the vaccines in use today
typically require multiple inoculations per year. Even with
multiple inoculations, the available vaccines do not achieve 100%
efficiency, and so other control measures e.g., the use of
acaracides, need to used in combination with the vaccine for full
control of the ticks.
[0012] Nevertheless, immunity to parasitic arthropods e.g., ticks,
is a highly desired form of parasite control. Indeed, what is
needed in the art is an effective vaccine that would provide
sustained and effective immunological response with a single
inoculation. Such a vaccine would avoid the problems associated
with acaricide resistance, chemical residues in food and the
environment, and the difficulty of breeding tick resistant species
for all animal production systems. Fortunately, as will be clear
from the following disclosure, the present invention provides for
this and other needs.
SUMMARY OF THE INVENTION
[0013] In one embodiment, the present invention provides a method
for stable transfection of Babesia parasites.
[0014] Other features, objects and advantages of the invention will
be apparent from the detailed description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 Illustrates an exemplary construct comprising bsd-gfp
fusion cassette in the expression site. In exemplary embodiments
the construct comprises a tick antigen gene, e.g., Bm86, fused to
the selectable marker bsd-gfp gene in the expression site.
[0016] FIG. 2 Illustrates plasmid p4-35 encoding luciferase under
the transcriptional control of the B. bovis ef-1.alpha.
promoter.
[0017] FIG. 3A and FIG. 3B demonstrates transfection of purified B.
bovis infected red blood cells (iRBC) by nucleofection and
electroporation with plasmid p4-35-luc. FIG. 3A. Comparison of
luciferase activity in lysates of B. bovis transfected with
plasmids p4-35-luc or pBS control, using nucleofection (4-35-Am;
pbs-am), or electroporation (4-35-BR; pbs-br); "nep-4-35" and
"nep-pbs" represent data from non transfected parasites. The
luciferase assays were performed 48 hours after transfection. RLU:
relative light units. The data in the columns represent the means
and standard deviations for RLUs obtained after two independent
replicates. FIG. 3B. B. bovis percentage of parasitized
erythrocytes (PPE) in cultures obtained 24 (blue) and 48 (crimson)
hours after transfection with plasmids p4-35-luc or pBS control,
using nucleofection (4-35-Am; pbs-am), or electroporation (4-35-BR;
pbs-br); "nep-4-35" and "nep-pbs" represent data from non
transfected parasites nucleofection or electroporation of iRBC. The
data in the columns represent the means and standard deviations for
the percentage of parasitized erythrocytes obtained after two
independent replicates.
[0018] FIG. 4 Structure of plasmid p4-35-ef-luc. The restriction
sites used for linearization (KpnI) and double digestion (KpnI and
NotI) are indicated with arrows.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0019] The term "parasitic arthropod" as used herein, refers to
arthropods that are parasitic on other species, typically feeding
on the blood of their host. Exemplary parasitic arthropods include,
but are not limited to ecto-parasitic arthropods e.g., ticks,
mites, flies, fleas, midges, etc., Parasitic arthropods are
potentially vectors of pathogens and thus can cause disease in
animals and man.
[0020] The term "tick" as used herein refers to small arthropod
arachnids that, along with other mites, constitute the order
Acarina. Ticks are ectoparasites, which live by hematophagy on the
blood of mammals, birds, and occasionally reptiles and amphibians.
Ticks are vectors of a number of diseases, including, but not
limited to babesiosis. Major families of ticks include the Ixodidae
or hard ticks, which have thick outer shells made of chitin, and
Argasidae or soft ticks, which have a membraneous outer surface. A
third family, Nuttalliellidae, contains one rare African species,
Nuttalliella namaqua. Soft ticks typically live in crevices and
emerge briefly to feed, while hard ticks will attach themselves to
the skin of a host for long periods of time. Tick bites look like
mosquito bites, but can also sometimes bruise or resemble a
bullseye.
[0021] The term "Babesia" as used herein refers to a genus of
parasitic protozoans including, but not limited to Babesia bovis,
Babesia microti, Babesia bigemina, Babesia divergens etc, which
affect vertebrates, including (though rarely) humans. Babesiosis is
the disease caused by the Babesia parasite, and typically occurs in
dogs, cattle, horses, and rodents. Babesia typically exhibit two
life cycles: one in the invertebrate host (typically ticks) and one
in the vertebrate host. Typical symptoms of babesiosis include
fever, anemia, fatigue, aches, chills, red urine, and possibly
death. Treatment is often unreliable or not available in the United
States, and many of the medications available may cause severe side
effects. Humans with babesiosis are usually treated with malaria
remedies.
[0022] The term " protective tick antigen" as used herein, refers
to an antigen that stimulates protective immunity to ticks.
Protective immunity may be exhibited in a number of forms including
but not limited to reduced or absent tick load due to failure of
ticks to attach, death of ticks after or during feeding, prevention
of ova production and death.
[0023] As used herein, the term "control" or "controlling" as in
e.g., the phrase: "controlling ticks" or "controlling tick
populations" or "controlling parasitic arthropods" or "controlling
parasitic arthropod populations", refers to any means for
preventing infection or infestation, reducing the population on
already affected organisms, or elimination of the population of
ticks or parasitic arthropods e.g. ticks, mosquitoes, lice fleas or
other species whose "control" is desired. Indeed, "controlling" as
used herein refers to any indica of success in prevention,
elimination, reduction or amelioration of a parasitic arthropod
population or parasitic arthropod problem.
[0024] The term "ameliorating" or "ameliorate" refers to any
indicia of success in the treatment or control parasitic
arthropods, including any objective or subjective parameter such as
abatement, or diminution of parasitic arthropod populations or an
improvement in a subject's physical well-being which may result
from decreased load of ticks feeding upon a subject. Amelioration
of parasitic arthropods e.g., ticks, can be based on objective or
subjective parameters; including the results of a physical
examination and/or total average parasite count.
[0025] The terms "isolated," "purified," or "biologically pure" as
used herein, refer to material that is substantially or essentially
free from components that normally accompany it as found in its
native state. In an exemplary embodiment, purity and homogeneity
are determined using analytical chemistry techniques such as
polyacrylamide gel electrophoresis or high performance liquid
chromatography. A protein that is the predominant species present
in a preparation is substantially purified. In one exemplary
embodiment, an isolated protective tick antigen nucleic acid is
separated from open reading frames that flank the protective tick
antigen gene and encode proteins other than the protective tick
antigen. The term "purified" denotes that a nucleic acid or protein
gives rise to essentially one band in an electrophoretic gel.
Typically, it means that the nucleic acid or protein is at least
85% pure, at least 90% pure, at least 95% pure, or at least 99%
pure.
[0026] The term "nucleic acid" as used herein, refers to a polymer
of ribonucleotides or deoxyribonucleotides. "Nucleic acid" polymers
typically occur in either single- or double-stranded form, but are
also known to form structures comprising three or more strands. The
term "nucleic acid" includes naturally occurring nucleic acid
polymers as well as nucleic acids comprising known nucleotide
analogs or modified backbone residues or linkages, which are
synthetic, naturally occurring, and non-naturally occurring, which
have similar binding properties as the reference nucleic acid, and
which are metabolized in a manner similar to the reference
nucleotides. Examples of such analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides,
peptide-nucleic acids (PNAs).
[0027] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid
Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98
(1994)).
[0028] The term "nucleic acid" also includes "recombinant nucleic
acid". The term "recombinant nucleic acid" as used herein refers to
a nucleic acid, not normally found in Nature that is typically
formed in vitro using modern techniques of molecular biology. Thus,
an isolated nucleic acid formed in vitro by ligating DNA molecules
that are not normally joined, is an exemplary "recombinant nucleic
acid".
[0029] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to naturally occurring amino acid
polymers and non-naturally occurring amino acid polymers as well as
amino acid polymers in which one or more amino acid residues is an
artificial chemical mimetic of a corresponding naturally occurring
amino acid.
[0030] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0031] Amino acids are referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
[0032] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0033] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art (see, e.g., Creighton, Proteins (1984)). Such conservatively
modified variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles.
[0034] The following eight groups illustrate some exemplary amino
acids that are conservative substitutions for one another: [0035]
1) Alanine (A), Glycine (G); [0036] 2) Aspartic acid (D), Glutamic
acid (E); [0037] 3) Asparagine (N), Glutamine (Q); [0038] 4)
Arginine (R), Lysine (K); [0039] 5) Isoleucine (1), Leucine (L),
Methionine (M), Valine (V); [0040] 6) Phenylalanine (F), Tyrosine
(Y), Tryptophan (W); [0041] 7) Serine (S), Threonine (T); and
[0042] 8) Cysteine (C), Methionine (M)
[0043] Macromolecular structures such as polypeptide structures are
described in terms of various levels of organization. For a general
discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3.sup.rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains.
Domains are portions of a polypeptide that form a compact unit of
the polypeptide and are typically 50 to 350 amino acids long.
Typical domains are made up of sections of lesser organization such
as stretches of .beta.-sheet and .alpha.-helices. "Tertiary
structure" refers to the complete three dimensional structure of a
polypeptide monomer. "Quaternary structure" refers to the three
dimensional structure formed by the noncovalent association of
independent tertiary units. Anisotropic terms are also known as
energy terms.
[0044] The term "label" as used herein, refers to a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and proteins for which antisera or monoclonal antibodies
are available.
[0045] As used herein a "nucleic acid probe or oligonucleotide" is
defined as a nucleic acid capable of binding to a target nucleic
acid of complementary sequence through one or more types of
chemical bonds, usually through complementary base pairing, usually
through hydrogen bond formation. As used herein, a probe may
include natural (i.e., A, G, C, or T) or modified bases (e.g.,
7-deazaguanosine, inosine, etc.). In addition, the bases in a probe
may be joined by a linkage other than a phosphodiester bond, so
long as it does not interfere with hybridization. Thus, for
example, probes may be peptide nucleic acids in which the
constituent bases are joined by peptide bonds rather than
phosphodiester linkages. It will be understood by one of skill in
the art that probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the
stringency of the hybridization conditions. In one exemplary
embodiment, probes are directly labeled as with isotopes,
chromophores, lumiphores, chromogens etc. In other exemplary
embodiments probes are indirectly labeled e.g., with biotin to
which a streptavidin complex may later bind. By assaying for the
presence or absence of the probe, one can detect the presence or
absence of the select sequence or subsequence.
[0046] Thus, the term "labeled nucleic acid probe or
oligonucleotide" as used herein refers to a probe that is bound,
either covalently, through a linker or a chemical bond, or
noncovalently, through ionic, van der Waals, electrostatic, or
hydrogen bonds to a label such that the presence of the probe may
be detected by detecting the presence of the label bound to the
probe.
[0047] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, over expressed, under expressed or
not expressed at all.
[0048] The term "promoter" as used herein refers to an array of
nucleic acid control sequences that direct transcription of a
nucleic acid. As used herein, a promoter comprises necessary
nucleic acid sequences near the start site of transcription, such
as, e.g., a polymerase II type promoter, a TATA element. In some
exemplary embodiments, a promoter also includes distal enhancer or
repressor elements, which can be, but are not necessarily located
as much as several thousand base pairs from the start site of
transcription. A "constitutive" promoter is a promoter that is
active under most environmental and developmental conditions. An
"inducible" promoter is a promoter that is active under
environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0049] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, a heterologous nucleic acid is
typically recombinantly produced, having two or more sequences from
unrelated genes arranged to make a new functional nucleic acid,
e.g., a promoter from one source and a coding region from another
source. Similarly, a heterologous protein indicates that the
protein comprises two or more subsequences that are not found in
the same relationship to each other in nature (e.g., a fusion
protein).
[0050] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed
operably linked to a promoter.
[0051] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (e.g., 80% identity, preferably 85%, 90%, or 95% identity
over a specified region) when compared and aligned for maximum
correspondence over a comparison window, or designated region as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. Such sequences are
then said to be "substantially identical." This definition also
refers to the compliment of a test sequence. Preferably, the
identity exists over a region that is at least about 25 amino acids
or nucleotides in length, or more preferably over a region that is
50-100 amino acids or nucleotides in length.
[0052] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0053] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0054] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. Using PILEUP, a reference sequence is
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps. PILEUP can be obtained from the GCG sequence analysis
software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids
Res. 12:387-395 (1984).
[0055] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information (ncbi.nlm.nih.gov/).
This algorithm involves first identifying high scoring sequence
pairs (HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) or 10, M=5, N4 and a comparison of both strands. For amino acid
sequences, the BLASTP program uses as defaults a wordlength of 3,
and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and
a comparison of both strands.
[0056] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0057] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0058] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0059] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60 .degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For high stringency hybridization, a positive signal
is at least two times background, preferably 10 times background
hybridization. Exemplary high stringency or stringent hybridization
conditions include: 50% formamide, 5.times.SSC and 1% SDS incubated
at 42 .degree. C. or 5.times.SSC and 1% SDS incubated at 65
.degree. C., with a wash in 0.2.times.SSC and 0.1% SDS at 65
.degree. C.
[0060] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides that they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cased, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37 .degree. C., and a wash in
1.times.SSC at 45 .degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency.
I. Introduction:
[0061] In one exemplary embodiment, the invention provides a method
for stably transfecting Babesia parasites. In one exemplary
embodiment, stably transfected Babesia parasites expressing a
heterologous nucleic acid encoding a tick protective antigen
provides an effective vaccine system conferring immunological
resistance to ticks.
[0062] In one exemplary embodiment, stably transfected Babesia
parasites expressing a heterologous nucleic acid encoding a tick
protective antigen are used to prepare live attenuated Babesia
parasites for vaccination.
II. Constructing an Expression Vector for Transfection and
Expression of Protective Antigens
[0063] A. General Recombinant DNA Methods
[0064] This invention utilizes routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
[0065] For nucleic acids, sizes are given in either kilobases (kb)
or base pairs (bp). These are estimates derived from agarose or
acrylamide gel electrophoresis, from sequenced nucleic acids,
and/or from published DNA sequences. For proteins, sizes are given
in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes
are estimated from gel electrophoresis, from sequenced proteins,
from derived amino acid sequences, or from published protein
sequences.
[0066] Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosphoramidite
triester method first described by Beaucage & Caruthers,
Tetrahedron Letts. 22:1859-1862 (1981), using an automated
synthesizer, as described in Van Devanter et. al., Nucleic Acids
Res. 12:6159-6168 (1984). Purification of oligonucleotides is by
either native acrylamide gel electrophoresis or by anion-exchange
HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149
(1983).
[0067] The sequence of the cloned genes and synthetic
oligonucleotides can be verified after cloning using, e.g., the
chain termination method for sequencing double-stranded templates
of Wallace et al., Gene 16:21-26 (1981).
[0068] B. Suitable Vectors
[0069] Development of stable targeted integration systems comprises
identification of strong promoters, introducing and expressing
foreign DNA, efficient selection markers, and the identification of
regions of the Babesia genome that will make good targets for
integration. The expression region of an exemplary expression
vector suitable for preparing stably transfected Babesia parasites
is shown in FIG. 1.
[0070] 1. Promoters
[0071] Exemplary promoters for construction of expression vectors
capable of expressing heterologus nucleic acids in stably
transfected Babesia cell lines include, but are not limited to
rap-1 (Suarez et al. (2004) Int. J. for Parasitology 34:1177-1184,
which is incorporated herein by reference). The sequence of the
rap-1 3' region used in the transfection vector are deposited in
GenBank under the following accession number: AF027149. In another
exemplary embodiment, the promoter comprises ef-1.alpha..
ef-1.alpha. is known in the art, see e.g., Suarez, C. E. et al
(2006) Int. J. for Parasitology 36:965-973 which is incorporated
herein by reference. The sequences for the of-1.alpha. Promoter and
of-la flanking regions that are used to construct an exemplary
transfection vector were deposited in GenBank under this accession
number: DQ322644.
[0072] Any suitable promoter can be used.
[0073] 2. Selective Markers
[0074] Exemplary selectable markers include, but are not limited to
the antifolate WR99210, puromycin and blasticidin. In one exemplary
embodiment, blasticidin is the selective marker used. However, any
suitable selectable marker known in the art can be used. Some
exemplary markers are disclosed in e.g., Wel, A. et al. Mol Biochem
Parasitol. 2004 March;134(1):97-104; Mamoun, C. B. et al., Proc
Natl Acad Sci U S A. 1999 Jul. 20;96(15):8716-20. Erratum in: Proc
Natl Acad Sci U S A 1999 Sep. 14;96(19):10944; Wang, P. et al., Mol
Biochem Parasitol. 2002 Aug. 7;123(1):1-10.
[0075] 3. Expressed Protective Antigens
[0076] Exemplary foreign antigens for eliciting protective immune
responses include, but are not limited to Tick (Boophilus
microplus, Dermacentor sp, etc.) antigens, Anaplasma marginale,
Babesia bigemina, etc, Bm86, Bm95.
[0077] In one exemplary embodiment, the protective tick antigen
present in the expression site of the expression vector is Bm86.
Bm86 is known in the art, see e.g., U.S. Pat. No. 5,587,311; and
Rand, K. N., et al. (1989) Proc Natl Acad Sci USA.
Dec;86(24):9657-61). An exemplary Bm86 nucleic acid sequence is
provided in GenBank as accession number: 132386. In one exemplary
embodiment, the protective tick antigen present in the expression
site of the expression vector is at least about 90% identical to
the sequence of Bm86 shown in GenBank accession number: I32386. In
other exemplary embodiments, the protective tick antigen present in
the expression site of the expression vector is at least about 91%
identical, at least about 92% identical, at least about 93%
identical, at least about 94% identical, at least about 95%
identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical, at least about 99%
identical, or at least about 100% identical to the sequence of Bm86
shown in GenBank accession number: I32386.
[0078] In an exemplary embodiment, the protective antigen is fused
to a gfp-bsd fusion open reading frame used in the transfection
vector shown in FIG. 1. In an exemplary embodiment, the gfp-bsd
fusion open reading frame used in the transfection vector shown in
FIG. 1 is amplified from vector pTracer-CMV/Bsd from InVitrogen bp
2576-3679. In some exemplary embodiments, gfp need not be present
in the vector.
[0079] In another exemplary embodiment Bm95 provides a protective
tick antigen. Bm95 is known in the art see e.g., Boue O., et al.
Exp Appl Acarol. 2004;32(1-2):119-28.
[0080] Other exemplary protective antigens include, but are not
limited to Bm91, which has regions of amino acid sequence
similarity to angiotensin converting enzymes. Calreticulin (CRT)
has been implicated in a wide variety of cellular processes and
affected numerous biological and pathophysiological conditions.
Therefore, CRT protein is an exemplary antigen against tick
infestations. Serine protease inhibitors (Serpins) have a potential
role of enzymatic regulatory inflammation mechanisms that are part
of tick feeding mechanisms. Thus, in an exemplary embodiment
Serpins are a protective tick antigen.
[0081] C. Cloning Methods for the Isolation of Nucleotide Sequences
Encoding Protective Tick Antigens
[0082] In general, the nucleic acid sequences encoding protective
tick antigens and related nucleic acid sequence homologs are cloned
from cDNA and genomic DNA libraries or isolated using amplification
techniques with oligonucleotide primers by methods well known in
the art. In an exemplary embodiment, protective tick antigen
sequence are typically isolated from cDNA libraries comprising DNA
sequences from Boophilus microplus. In one exemplary embodiment,
antigens not normally involved in acquired resistance are isolated,
cloned into an appropriate expression vector, transfected into
Babesia, and are thus used to induce anti-tick immunity. In one
exemplary embodiment, gut proteins e.g., Bm86, comprise protective
tick antigens.
[0083] In an exemplary embodiment, DNA sequences which are similar
to and/or homologous with the DNA coding for the tick protective
antigens e.g., from Boophilus microplus, can be used to identify
DNA sequences from other tick species by constructing cDNA or
genomic DNA libraries for the other tick species and hybridizing
Boophilus microplus DNA fragments to those libraries, and purifying
recombinant organisms containing the DNA sequences hybridizing to
the homologous genes.
[0084] D. Assays for Discovering Alternative antigens
[0085] Antigens not normally involved in acquired resistance are
exemplary protective tick antigens that can be used to induce
anti-tick immunity using transgenic babesia vaccines. In an
exemplary embodiment, these antigens are obtained from tick gut
absorptive surface. Sera obtained from immunized animals can be
used to identify antibody-reactive components of the
resistance-inducing extract. Tick gut absorptive surface antigen
glycoconjugates can be identified by lectin blotting, using a
series of probes with different carbohydrate specificities by
methods known in the art see e.g., Wikel S K. Vet Parasitol. 1988
September;29(2-3):235-64.
[0086] In an exemplary embodiment, antibody-reactive components of
the resistance-inducing extract are reverse cloned i.e., protein
sequence is used to deduce nucleic acid sequence, so that probes
can be synthesized and hybridized to libraries to obtain the gene
encoding a new protective tick antigen.
III. Transfection Systems
[0087] The tick borne Babesia parasites are useful as live
attenuated vaccines for control of Babesia parasites. Additionally,
in an exemplary embodiment, Babesia parasites provide a convenient
vector for the expression of antigens protective against ticks and
other parasitic arthropods. Indeed, in one exemplary embodiment,
vaccination with live attenuated Babesia parasites which have been
transfected with protective tick antigens e.g., Bm86, Bm95,
provides vaccine effective to protect the vaccinated animal against
ticks as well as against Babesia.
[0088] In one exemplary embodiment, stable transfection of Babesia
bovis is useful for introducing and expressing transgenes in B.
bovis parasites that are used for preparing live Babesia attenuated
strains that express foreign genes and thus, the foreign genes
produce antigens that function as vaccines. In another exemplary
embodiment, stable transfection of Babesia bovis is is used to
introduce and express markers that identify vaccinated animals.
[0089] Transfection of other parasitic organisms which affect
vertebrate hosts and which require an intermediate arthropod vector
can be used to prepare transgenic strains of the parasite, which
express protective antigens against the arthropod. Exemplary
parasitic organisms include but are not limited to Plasmodium, see
e.g., Balu, B., and Adams, J. H. (2007) Int J Parasitol.
Jan;37(1):1-10; and et al. (2004) Methods Mol Biol. 270:263-76.
[0090] A. Transient Transfection
[0091] In an exemplary embodiment, Babesia strains are transiently
transfected with genes encoding antigens protective against
parasitic arthropods e.g., ticks. Transient transfection is known
in the art, see e.g., Suarez et al. (2004) Int. J. for Parasitology
34:1177-1184.
[0092] B. Stable Transfection
[0093] In an exemplary embodiment, Babesia strains are stably
transfected with genes encoding antigens protective against
parasitic arthropods e.g., ticks. In an exemplary embodiment,
expression vectors as disclosed in section II (above) are
introduced into either Babesia infected erythrocytes or free
merozoites.
[0094] In an exemplary embodiment, transfection is performed with a
conventional electroporation system (BioRad GenePulser II). In
another exemplary embodiment, transfection is performed with
nucleofection technology (Amaxa). Typically, conventional
electroporation is more effective for transfection of larger
amounts of plasmid (100 .mu.g range) whereas typically,
nucleofection is more efficient for transfecting much smaller
amounts (2 .mu.g range), of circular or linearized plasmids.
[0095] In an exemplary embodiment, targets for gene substitution
upon transfection are genes that are not needed for development of
the parasite during its growth in in vitro cultures, although other
genes may also be targeted. In one exemplary embodiment stable
transfection is achieved using rap-1 and ef-1.alpha. genes as
targets through homologous recombination. As is known in the art,
double chain cuts delivered e.g., by restriction endonucleases, to
homologus sequences located on the targeting (expression) vector
direct recombination to sites in the genome homologus with the cut
vector DNA(see e.g.,, Szostak, J. W. et al. (1983) Cell 33:25-35).
In other exemplary embodiments, appropriate targets are identified
using the B. bovis genome data and characterization of
stage-specific transcripts. In one exemplary embodiment, 18s
ribosomal units serve as target sequences, since the three B. bovis
18s ribosomal units can be differentially transcribed among
distinct life stages of the parasite, with one of the genes not
showing transcripts during in vitro merozoite culture stages. Thus,
this region of the B. bovis genome can be targeted for integration
without compromising parasite viability. In one exemplary
embodiment B. bovis 18s ribosomal genes are used for preparing
stable transfection constructs targeted to this region of the
genome. In another exemplary embodiment, non-targeted integration
is achieved using in transposons and pantropic retroviral
vectors.
[0096] IV. Vaccinating Livestock
[0097] Live attenuated Babesia vaccines are known in the art see
e.g., Callow, L. L. et al. (1997) Int. J. for Parasitology
27(7):747-767; Anonymous. (1984) Immunization against bovine
babesiosis. In: Ticks and Tick-borne Disease Control. A Practical
Field Manual, Vol. II Chapter 10, pp 388-443 Food and Agriculture
Organization, Rome; and de Waal, D. T., and Combrink, M. P. (2006)
Vet Parasitol. May 31;138(1-2):88-96. Epub 2006 Febtuary 28.
Review.
[0098] In one exemplary embodiment, Babesia parasites stably
transfected with a heterologus nucleic acid capable of expressing a
protective tick antigen, are used to prepare a live attenuated
Babesia vaccine, by methods known in the art.
[0099] The live attenuated Babesia vaccine is administered to an
animal, e.g., cattle, horse, dog, deer, goats, sheep, cats, pigs
etc., in need thereof, by methods known in the art. In the animal,
the protective tick antigen in the transgenic Babesia is expressed,
thereby stimulating a protective immune response against the tick
antigen and thus providing protective immunity against the tick
ectoparasite.
[0100] In one exemplary embodiment, vaccination with transgenic
Babesia induces permanent immunity, since animals infected with
Babesia remain infected for life.
[0101] V. Testing for Immunization
[0102] In one exemplary embodiment the expression of molecular or
antigenic tags on the expression vector are used for the
identification and discrimination of vaccinated animals in the
field. In an exemplary embodiment, the presence of antibodies or
parasites in field animals is analyzed to determine whether these
animals were naturally infected or previously vaccinated, for
treatment or epidemiological purposes. Because transfected Babesia
vaccine strains contain a gene that is not present in wild type
strains; animals that have received the vaccine are readily
identified by detection of the not present in wild type strains. In
one exemplary embodiment, the gene not present in wild type strains
is detected by PCR amplification. In another exemplary embodiment,
the gene not present in wild type strains is expressed in the
animal as an antigen which stimulates antibody production. Thus,
vaccinated animals will elicit antibody responses against these
expressed antigens, and these animals can also be identified using
serological reactions, such as ELISA.
[0103] In another exemplary embodiment, the ability to detect,
e.g., with PCR or immunological methods, animals vaccinated with
transfected Babesia strains is useful for determining whether
vaccine breakthroughs are due to the vaccine strain that reverted
to virulence (not uncommon), or lack of protection of the vaccine
strain against some virulent field strain due, for instance to
antigenic variation.
[0104] In still another exemplary embodiment, vaccination with
transfected Babesia vaccine strains permits tracking of the
transmission of the vaccine strain to ticks or to other naive
animals via tick transmission. In one exemplary embodiment, he
inclusion of the green fluorescent protein (gfp) gene in the
transfection construct allow easy tracking by fluorescence
microscopy of the localization of the vaccine strain in tick
tissues, and in non-vaccinated animals. In other exemplary
embodiments, tracking is facilitated by inclusion of any other
expression of unique antigens in the vaccine strain, e.g., by PCR,
immunological methods, immunofluorescence and/or in situ
hybridization.
VI. Other Uses for A Babesia Stable Transfection System
[0105] The full sequence of the Babesia genome is now available,
see e.g., Journal of Medical Entomology, Volume 43, Number 1,
January 2006, pp. 9-16(8) and Brayton, K. A. et al. (2007) PloS
Pathog. 3:1401-1413. Many of the sequenced genes are of unknown
function. In one exemplary embodiment, a Babesia stable
transfection system as disclosed herein, is useful for functional
characterization of Babesia genes by permitting the construction of
knock out strains. Knock out analysis of gene function is well
appreciated by those of skill in the art. In one exemplary
embodiment, a Babesia stable transfection system as disclosed
herein, is useful for the characterization of Babesia
promoters.
[0106] Babesia bovis and Babesia bigemina are important causative
agents of bovine babesiosis in tropical and subtropical regions of
the world. Babesia divergens is more common in temperate climates.
Babesiosis was a significant problem in the southern US until the
1940's when it was controlled by eradication of the tick vectors by
intensive acaricide dipping of cattle. However, the tick vectors
are present in a buffer zone along the Rio Grande, in Mexico, and
in US territories, and pose the threat of continual reemergence
into the US as evidenced by occasional outbreaks of babesiosis in
the border region. Emerging acaracide resistance of vector ticks in
Mexico is a significant concern, since re-introduction of
babesiosis into the US likely will occur via infected ticks. It is
estimated that the first year cost of controlling vector ticks
alone should they be introduced into the U.S. is over $1.3 billion.
There is currently no babesial vaccine licensed for use in the
U.S., and development of a vaccine is a high priority.
[0107] Thus, in one exemplary embodiment, a Babesia stable
transfection system as disclosed herein, is useful for rational
identification of subunit vaccine candidates for the development of
a vaccine that is acceptable for use in the U.S. Indeed, in one
exemplary embodiment, a Babesia stable transfection system is used
to discover useful protective antigens which can be used directly
as vaccines to protect against Babesia infection. In another
exemplary embodiment, genes known to express useful protective
antigens are cloned into an appropriate expression vector and
stably transfected into Babesia for preparation of live attenuated
vaccines to protect against further Babesia infection.
[0108] In another exemplary embodiment, the ability to stably
transform B. bovis parasites provides a means for better
understanding the biology of Babesia parasites by providing means
for understanding parasite associated determinants of virulence,
tick transmission, and immunity
[0109] The following examples are offered to illustrate, but not to
limit the invention.
EXAMPLES
Example 1
[0110] The following example illustrates exemplary methods for
obtaining Babesia trasfectants for use in the preparation of
vaccines that protect against ticks and that may also protect
against Babesia.
Materials and Methods
[0111] Parasites: The Mol biological clone of B. bovis was derived
by limiting dilution of the Mexico strain as described (Rodriguez
et al, (1983) Infect Immun. 42:15-18; Hines et al. (1989) Mol.
Biochem. Parasitol. 37:1-10), and was maintained as a cryopreserved
stabilate in liquid nitrogen (Palmer et al., (1982) Parasitology.
84:567-571). Parasites were grown in long term microaerophilous
stationary-phase culture by previously described techniques (Levi
and Ristic, (1980) Science. 207:1218-1220; and Hines et al., (1989)
supra). B. bovis purified merozoites were obtained from 8 to
10-flask expansion of B. bovis typically containing between 30-40%
infected red blood cells (iRBC) as determined by microscopic
counting of Giemsa stained slides, as described previously (Hines
et al. (1992), Mol. Biochem. Parasitol. 55:85-94).
[0112] Plasmid constructs: Plasmid p4-35 encoding luciferase under
the transcriptional control of the B. bovis ef-1.alpha. promoter
(FIG. 2) was described previously (Suarez, C. E., et al., (2006)
Int J. Parasitol. 36, 965-973, which is incorporated herein by
reference). Plasmids were purified using the Qiagen endotoxin-free
maxiprep kit (Qiagen Inc., Calif.) following the manufacturer's
instructions.
[0113] Amaxa nucleofection of purified Babesia bovis merozoites:
Varying amounts of plasmid (2 to 100 .mu.g) suspended in 100 .mu.l
of Amaxa Nucleofactor reagent (Amaxa ByoSystems Inc.) were added to
10 .mu.l of free merozoites (.about.2.times.10.sup.6 free
merozoites) mixed to avoid bubbles, and transferred to an 0.2 cm
Amaxa electroporation cuvette. The cuvette was then placed in the
Amaxa nucleofection device and nucleofected according to
manufacturer's instructions using the Amaxa program v-024 in
conjunction with the "Plasmodium" buffer. Immediately after
nucleofection the cuvette contents were transferred to a culture
well containing 1.1 ml of Babesia culture media with 10% normal
RBCs. Transfected parasites were cultured in 24 well culture plates
as described previously.
[0114] Electroporation: Electroporation was performed in a Gene
Pulser II apparatus (BioRad) using 0.2cm cuvettes containing filter
sterilized cytomix buffer (120 mM KCl, 0.15 mM CaCl.sub.2, 10 mM
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM
EGTA, 5 mM McCl.sub.2, final pH 7.6) at a final volume of 100
.mu.l. Typically, 1-2.times.10.sup.6 merozoites per cuvette were
used for electroporation, except when indicated otherwise.
Following electroporation, merozoites were cultured as described
before for nucleofection.
[0115] 2. 5 Luciferase assays: Luciferase analysis was performed as
described previously (Suarez et al., (2004) Int. J. Parasitol. 34,
1177-1184, which is incorporated herein by reference) using
Promega's LAR II detection reagent at room temperature along with a
Turner Designs TD-20/20 Tube Luminometer for a 10 second
integration. For each set of luciferase assays, 2 .mu.l of a
10.sup.-6 dilution of Promega's QuantiLum Recombinant Luciferase
diluted in 1.times. Passive Lysis Buffer was assayed as a
standard
[0116] Results and Discussion
[0117] The above disclosed experiments compare the relative
efficiency of the BioRad GenePulser II electroporation method
(Suarez et al. (2006) supra) and the Amaxa nucleofection method
(Balu et al. (2007) Int. J. Parasitol. 37:1-10; Janse et al.,
(2006) Mol. Biochem. Parasitol. 145:60-70) for transfecting
purified merozoites or infected erythrocytes with variable
quantities of plasmid. p4-35-luc containing luciferase as a
reporter gene under the control of the ef-1.alpha. promoters in the
5' region, and the 3' region of rap-1 (FIG. 1) (Suarez et al.,
(2006) supra). Thus, plasmid p4-35-luc or control pBS was first
transfected into 10.sup.8 infected red blood cells (iRBC)
containing about 30% iRBC parasitemia, with the Amaxa nucleofector
to determine whether this device was suitable for the introduction
of foreign plasmid DNA into infected red blood cells (iRBC) using
the nucleofection settings that we previously determined for the
transfection of purified merozoites (Suarez et al., 2007,
manuscript submitted). The results of the Amaxa nucleofection of
iRBC compared with BioRad electroporation performed on identical
number of parasites using 100 .mu.g of transfected plasmid are
shown in FIG. 3A and FIG. 3B. The luciferase values, measured 48
hours after transfection shown in FIG. 3A indicate that
nucleofection also transfers plasmid DNA into iRBC, although it
appears to be less efficient than electroporation at least in the
settings tested in this experiment. In FIG. 3B shows the percentage
of parasitized erythrocytes (PPE) in the first and second day after
transfection. No differences in parasite viability were detected
with this method when electroporation and nucleofection were
compared. However, transfection resulted in decreased viability
when the PPE of the transfected (either nucleofected or
electroporated) with the non-transfected controls (nep 4-35 and
nep-pBS) were compared.
[0118] The efficiency of both transfection methods for transferring
plasmid into iRBC was compared, as described above or into 10.sup.6
purified merozoites using either a large quantity of plasmid (100
.mu.g) or a smaller quantity of plasmid (2 .mu.g) . Luciferase was
measured at 24, 48 and 72 h, and the percentage of parasitized
erythrocytes was also calculated as a parameter of viability.
Parasites transfected with control plasmid pBS did not produce any
significant luciferase activity. Overall, the data indicates that
the peak of luciferase expression in transfected parasites occurs
at 24 hours in all cases, and that the most efficient method of
transfection tested in this study was electroporation of infected
erythrocytes with 100 .mu.g of plasmid, followed by nucleofection
with 2 .mu.g of plasmid. The data also confirms previous
observations that nucleofection is more efficient than
electroporation for transfecting small quantities of plasmids (2
.mu.g range), whereas the inverse is true for transfection of
larger quantities (100 .mu.g range). Data in FIG. 4 shows a
comparison of actual luciferase expression values expressed as RLU,
in conjunction with the percentage of parasitized erythrocyte at
24, 48 and 72 hours after transfection. Data indicates that while
the PPE increases, the RLU drops rapidly, consistent with the
transient nature of the transfection. Similar growth patterns were
observed in the four systems under study, therefore suggesting that
a significant number of parasites remain viable after transfection
regardless of the method used.
[0119] In summary, we demonstrated the feasibility of transfecting
B. bovis infected erythrocytes by the method of nucleofection, and
we also compared iRBC and purified merozoite transfection methods.
The most efficient transfection method tested in this study uses
100 .mu.g of plasmid on infected erythrocytes, but a very high rate
of efficiency was also obtained using Amaxa nucleofection, which is
the method of choice when using small quantities of plasmid for
electroporation. This scenario is likely to occur when attempting
transfection of restriction-enzyme linearized constructs to
facilitate double cross-over integration mechanisms. Overall, these
results illustrate stable transfection of exogenous DNA into
Babesia parasites.
Example 2
[0120] The following example illustrates an exemplary method for
the preparation Babesia parasites stably transfected with a
heterologous nucleic acid. In one exemplary embodiment, stable
transfection systems for Babesia are useful for functional analysis
of the recently sequenced Babesia bovis genome (Brayton K. A., et
al. supra). In addition, the ability stably transfect Babesia cells
allows one of skill to tknock out specific genes and to express
transgenes. In exemplary embodiments stably transfected Babesia
parasites are exploited for the development of attenuated Babesia
vaccine strains co-expressing foreign genes as immunogens. In some
exemplary embodiments, markers are also introduced into stable
transfectant cell lines that can be used to identify vaccinated
animals.
[0121] FIG. 4 illustrates the structure of plasmid p4-35-ef-luc.
The restriction sites used for linearization (KpnI) and double
digestion (KpnI and NotI) are indicated with arrows.
[0122] Nucleofection and electroporation systems for DNA
transfections as disclosed above in Example 1, were used to
transfect plasmid p4-35-ef-luc into Babesia merozoites. Plasmids
were either circular, singly digested with KpnI or doubly digested
with KpnI and NotI.
[0123] For restriction enzyme digestion, 10 .mu.g of plasmid was
digested with the indicated restriction enzyme (KpnI or NotI)
following manufacturer's instructions. Both linearized (KpnI) or
double digested (KpnI and NotI) plasmids were heated to 65.degree.
C. to inactivate the enzyme and then ethanol precipitated with
sodium chloride and suspended to 2 .mu.g/5 .mu.l in sterile water.
The completeness of digestion of plasmid DNA was confirmed by gel
electrophoresis.
[0124] Plasmid p4-35-ef-luc was used to analyze if one transfection
system would be more or less effective for transfection of
2.varies.g of circular plasmid, plasmid linearized with KpnI, or
plasmid double digested with KpnI and NotI. The results indicate
that luciferase expression from linear and the double digested
plasmids were much lower when compared with circular plasmid using
either transfection system. Nucleofection was markedly more
efficient for transfecting restriction-digested DNA. No significant
difference in post-transfection percentage parisitized erythrocytes
(PPE) was observed among the three groups.
[0125] The overall decrease in luciferase expression in the linear
and the double-digested DNA when compared to circularized plasmid
may be due to the increased stability of circular plasmid in the
presence of parasite endogenous nucleases, a possible increased
efficiency for transfection of the circular plasmid DNA, or a
combination. In summary, results suggest that nucleofection is a
method of choice for Babesia bovis merozoite transfection, when
introducing smaller quantities of plasmid, or restriction digested
DNA.
[0126] Example
[0127] The following example illustrates stable integration of
plasmid DNA into the genome of Babesia bovis using
blasticidin/blasticidin deaminase selection.
[0128] Blasticidin/blasticidin deaminase selection has been shown
to work with other protozoa. Furthermore no known bsd gene is
present in the B. bovis genome. Thus, the occurance of resistant
strains that are not transfected with the plasmid is reduced.
Blasticidin proved to be an efficient inhibitor of the growth of B.
bovis with an IC.sub.50 of .about.0.4 .mu.g/ml, a drug
concentration comparable to the IC.sub.50 (0.35 .mu.g/ml)
calculated for Plasmodium falciparum parasites. In addition, a
gfp/bsd fusion gene was used to incorporate an additional marker of
expression. With this construct, blasticidin resistant parasites
emerged relatively quickly after selection. The
blasticidin-resistant transfected B. bovis parasites are able to
grow in high concentrations of blasticidin with growth curves that
are similar to non-selected parasites in the absence of
blasticidin. Importantly, expression of the transfected
gfp-bsdfusion protein in B. bovis is not toxic for the parasites,
which have been able to strongly express the gfp-bsd protein at
least nine months after electroporation.
[0129] The plasmid pgfp-bsd-ef was introduced into B. bovis
infected erythrocytes by electroporation using several experimental
strategies. These included circular, linearized and double digested
plasmid in the presence or absence of the restriction enzyme NotI.
It was previously reported that addition of NotI, or other
restriction enzymes, in the transfection mix results in a 29-46
fold increase in transfection efficiencies, a technique known as
restriction enhanced mediated integration (REMI) (Black et al.,
(1995) Mol. and Biochem Parasitol. 74: 55-63). In our experiments,
transfectants able to grow in the presence of blasticidin were
derived both in the presence and the absence of NotI. These
transfectants are able to consistently express gfp-bsd for 9
months, consistent with stable expression. This suggests that the
addition of NotI was not required for producing stable
transformants.
[0130] Stable integration of the gfp-bsd gene into the B. bovis
genome is supported by the following observations: 1) sustained
growth in culture media containing concentrations of blasticidin
that are otherwise inhibitory for wild type Babesia, 2) detection
of fluorescence in transfected parasites more than 9 months after
electroporation, 3) inability to recover plasmid in plasmid rescue
experiments, 4) identification of gfp-bsd transcripts and
expression of a gfp-bsd fusion protein in transfected merozoites,
and 5) evidence of integration into the of-1.alpha. .quadrature.
locus in Southern blots and flanking PCR amplicons. Taken together,
the experimental evidence demonstrates stable transfection of B.
bovis parasites.
Materials and Methods
Parasites
[0131] The Mo7 biological clone of B. bovis was derived by limiting
dilution of the Mexico strain as described (Rodriguez et al, (1983)
Infect Immun.42: 15-18; Hines et al. (1989) Mol. Biochem.
Parasitol. 37, 1-10), and was maintained as a cryopreserved
stabilate in liquid nitrogen (Palmer et al., (1982) Parasitology.
84: 567-571). Parasites were grown in long term microaerophilous
stationary-phase culture by previously described techniques (Levi
and Ristic (1980) Science. 207: 1218-1220; Hines et al., 1989
supra).
Blasticidin Inhibition
[0132] B. bovis parasites of the Mo7 strain were cultured in
.about.1 ml cultures in 24 well plates containing 10% bovine red
blood cells. Triplicate wells were cultured with or without
increasing amounts of blasticidin added to the culture media at
0.15, 0.3, 0.6, 1.2, 1.5, 2.4, and 4.8 .mu.g/ml, prepared from a 5
mg/ml stock solution of blasticidin (Invitrogen) in sterile water,
for three consecutive days. Culture media was replaced daily. The
percentage of parasitized erythrocytes (ppe) was estimated by
microscopic counting of Diff-Quik (Dade Behring) stained slides as
described (Suarez and McElwain, (2008) Exp Parasitol. 118:
498-504). Percentage B. bovis inhibition was calculated by dividing
the mean ppe in triplicate blasticidin treated wells by the mean
ppe in triplicate control wells grown in the absence of
blasticidin, and multiplying by 100.
Plasmid Constructs
[0133] Plasmid pgfp-bsd-ef (FIG. 2 for diagrammatic representation)
was derived from previously described plasmid p40-15-luc (Suarez et
al., 2006 supra). The first 673 bps of the 5' region of the B.
bovis ef-1.alpha. orf were amplified from B. bovis Mo7 genomic DNA
using 6 primers Xho-EF-orf-F1 (5'-ctg acg ctc gag atg ccg aag gag
aag act cac-3' SEQ ID NO:1) and Xho-EF orf-R1 (5'-cag ctg ctc gag
atc tga tca agg gcc tcg acc-3' SEQ ID NO:2). The resulting 673 bp
amplicon was digested with XhoI, and cloned into the XhoI site of
plasmid p40-15-luc (Suarez et al., 2006, supra). The plasmid
obtained was designated p40-15-luc-XhoI-ef-5'. The 673 bp of the 3'
half of the B. bovis ef-1.alpha. orf were amplified from B. bovis
Mo7 genomic DNA using primers Bam-EF-orf-F2: (5'-gca tcg gga tcc
gga acc ccc aaa gag gcc cgt tg-3') and Bam-EF-orf-R2: (5'-cta gca
tcc tct tag cag cct ttt ggg cag ac-3'). The resulting 673 bp
amplicon was digested with BamHI and cloned into the BamHI site of
plasmid p40-15- luc-XhoI-ef-5'. This plasmid was designated
p40-15-luc-XhoI-ef-5'-ef-3 `BamHI. The gfp bsd fusion gene
amplified from plasmid pTracer (Invitrogen) using primers
Tracer-gfp-EcoI-F (5`- cgt cgt gaa ttc atg gcc tcc aaa gga gaa gaa
c-3' SEQ ID NO:3) and Tracer-gfp-EcoI-R (5'- taa tgt gaa ttc gcc
ctc cca cac ata acc aga g-3' SEQ ID NO:4) and digested with EcoRI
was cloned into EcoRI-alkaline phosphatase treated plasmid
p40-15-luc-XhoI-ef-5'-ef-3'BamHI, replacing the Luc gene. Finally,
the resulting plasmid was modified to include an additional NotI
site 3' to the XhoI insert by inserting the olinucleotide
Apa-Not-Apa (5'-gcg gcc gcg gcc-3 SEQ ID NO:5) into the ApaI site,
and the resulting construct was designated pgfp-bsd-ef. Plasmids
were purified using the Qiagen endotoxin-free maxiprep kit (Qiagen
Inc., Calif.) following the manufacturer's instructions. For NotI
restriction enzyme digestion, 10 .mu.g of plasmid was digested with
the restriction enzyme following manufacturer's instructions. NotI
digested plasmids were heated to 75.degree. C. for 20 minutes to
inactivate the enzyme and then ethanol precipitated with sodium
chloride and suspended in 5 .mu.l of sterile water. The
completeness of digestion of plasmid DNA was confirmed by gel
electrophoresis.
[0134] Electroporation of Babesia bovis infected erythrocytes and
drug selection B. bovis infected erythrocytes from an 8 flask
expansion were centrifuged at 400.times. g in a table-top
centrifuge for at 5 minutes to pellet the erythrocytes. The cell
pellet was washed one time in filter sterilized cytomix buffer (120
mM KCl, 0.15 mM CaCl.sub.2, 10 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4
pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl.sub.2, final pH
7.6) and centrifuged again as before. Electroporation was performed
in a Gene Pulser II apparatus (BioRad) using 0.2 cm cuvettes
containing 62.5 .mu.l filter sterilized cytomix plus the digested
plasmid and 37.5 .mu.l of washed B. bovis infected red blood cells
to a final volume of .about.100 .mu.l. Typically,
1-2.times.10.sub.8 infected erythrocytes per cuvette were used for
electroporation. The electroporator settings used were 1.2 kv/25
.mu.F/200 ohms (Suarez and McElwain, 2008, supra). The experimental
protocol for transfection is summarized in Table I.
[0135] Briefly, infected erythrocytes were electroporated with 20
.mu.g of circular plasmid or plasmid linearized with NotI, in the
presence or absence of NotI enzyme in the cuvette (Black et al.,
1995 supra). Control cultures contained either circular or NotI
linearized plasmid without NotI enzyme. In total, this resulted in
12 different experimental parameters (Table I). Following
electroporation, infected erythrocytes were cultured as described
above. For blasticidin selection, a 5mg/ml blasticidin stock
solution was prepared and stored at -80.degree. C. Blasticidin was
added to the culture media at concentrations as indicated in the
Results section, starting 24 hrs after electroporation. The ppe was
calculated daily as described above. Parasites growing in moderate
blasticidin concentration in culture wells from experimental
parameters 1 and 2 (Table 1) were combined and subjected to high
concentration blasticidin selection to obtain parasite line 1-2-124
(see below, Results).
TABLE-US-00001 TABLE 1 Experimental protocol for electroporation of
plasmid pgfp-bsd-ef into the Mo7 strain of B. bovis. NotI added
Actual Time Blasticidn to voltage constant Selection Plasmid and
format cuvette (Kv) (sec) (.mu.g/ml) 1. Circular pgfp-bsd-ef - 1.31
0.32 0.6* 2. Circular pgfp-bsd-ef - 1.24 0.26 0.3* 3. Circular
pgfp-bsd-ef + 1.26 0.52 0.6* 4. Circular pgfp-bsd-ef + 1.31 0.38
0.3 5. NotI-digested pgfp-bsd-ef - 1.33 0.28 0.6 6. NotI-digested
pgfp-bsd-ef + 1.26 0.58 0.3* 7. NotI-digested pgfp-bsd-ef + 1.31
0.34 0.6* 8. NotI-digested pgfp-bsd-ef + 1.31 0.32 0.3* 9.
pBS-circular - 1.24 0.3 0.6 10. pBS-circular - 1.26 0.56 0.3 11.
Linear pBS (NotI digested) - 1.31 0.32 0.6 12. Linear pBS (NotI
digested) - 1.28 0.5 0.3 *Experiments in which
blasticidin-resistant transfectants were identified.
Characterization of Putative Stably Transfected Parasite Line
1-2-124
[0136] Following transfection and blasticidin selection, a parasite
line designated 1-2-124 (see below, Results) was analyzed in detail
to confirm correct expression of the gfp-bsd fusion protein and
integration into the chromosome. This parasite line was followed
temporally using fluorescence microscopy, reverse transcriptase
PCR, and Western blot to confirm continuous expression of the
gfp-bsd gene. Southern blots, direct PCR and sequence analysis were
used to examine and genetically characterize chromosomal
integration.
Genetic Characterization
[0137] B. bovis merozoite total RNA from parasite line 1-2-124 (see
below, Results) was extracted from in vitro cultures by the
standard TRIzol (Life Technologies) procedure as described
previously (Suarez et al. (2003) Mol Biochem Parasitol.
127:101-12), and treated with RNAse-free DNAse (Ambion). For RT
PCR, Superscript First Strand Synthesis System kit (BRL-Invitrogen)
was used to generate cDNA with 1 .mu.g of total RNA from cultured
parasites. Reactions were carried out as per the manufacturer's
recommendation for first-strand synthesis using an Oligo(dT)
primer. The full size gfp-bsd orf was amplified either from genomic
DNA or from cDNA using the Gold Taq Polymerase kit (Applied
Biosystems) and the specific primers Tracer-gfp-EcoI-F and
Tracer-gfp-EcoI-R (see above). Products of RT-PCR were cloned into
vector pCR 2.1 (Invitrogen) and sequenced. Genomic DNA was
extracted from cultured merozoites by the standard
phenol-chloroform procedure. For Southern blot analysis, genomic
DNA from B. bovis merozoites was digested with restriction enzyme
BglII, electrophoresed, transferred to ZetaProbe Nylon membranes,
and hybridizations carried out as previously described (Suarez et
al., 2003, supra). Digoxigenin-labeled probes representing the
complete gfp-bsd and ef-1.alpha. orf's were prepared by PCR
amplification using a PCR Dig-Probe Synthesis kit as recommended by
the manufacturer (Boehringer-Roche). The gfp-bsd probe was prepared
by PCR amplification of plasmid pTracer (InVitrogen) with primers
gfp-bsd-f (5- atg gcc tcc aaa gga gaa gaa c-3' SEQ ID NO:6), and
gfp-bsd-r (5'- gcc ctc cca cac ata acc aga g -3' SEQ ID NO:7). The
r5-r6 amplicon was obtained by PCR amplification of genomic DNA of
the parasite line 1-2-124 with the set of primers ef-rev5 (5'-cat
atc aag ctt ctt taa cgg gat gac ata tat g-3' SEQ ID NO:8) and
ef-rev6 (5'-gac cat aag ctt agt aaa cga tag aac aga cta ag-3'SEQ ID
NO:9). The amplicon was cloned into plasmid vector pCR 2.1
(Invitrogen) and sequenced in full by standard techniques.
Immunoblot Analysis
[0138] To confirm the expression of correctly sized gfp-bsd fusion
protein, merozoites of parasite line 1-2-124 were subjected to
SDS-PAGE and analyzed by western blot. Immunoblots were performed
as described previously (Suarez et al., 2003 supra) using anti-GFP
antibody (Invitrogen) at a dilution of 1:104, and goat anti
rabbit-immunoglobulin peroxidase conjugate (Life Biosciences).
Results
[0139] Growth of Babesia bovis is Inhibited by Blasticidin in In
Vitro Cultures:
[0140] Previous unsuccessful attempts to stably transform B. bovis
utilized DHFR-induced resistance to pyrimethamine or WR99210 for
selection of transfected lines (Gaffar et al., 2004 Mol. Biochem.
Parasitol. 133:209-19). Natural resistance to these drugs developed
rapidly in culture (Gaffar et al., 2004, supra). To determine
whether B. bovis growth could be inhibited with blasticidin, and to
estimate the concentration of blasticidin appropriate for
establishing a blasticidin/blasticidin deaminase (bsd) transfection
system, B. bovis Mo7 strain parasites were cultured in the presence
of varying concentrations of blasticidin ranging from 0 to 5
.mu.g/ml in triplicate wells. The percentage of parasitized
erythrocytes (ppe) in each well was determined for the first three
days after splitting the cultures in each well to a starting ppe of
0.5%. The results obtained in the third day of blasticidin
selection indicate that B. bovis is sensitive to blasticidin. No B.
bovis infected erythrocytes could be detected in stained slides
after three days in culture using a blasticidin dose of 5 .mu.g/ml.
The 50% inhibitory concentration of blasticidin (IC.sub.50) after
three days of drug selection was determined to be 10.about.0.4
.mu.g/ml, and a concentration of blasticidin in the 0.64 -1.25
.mu.g/ml range resulted in negligible parasite growth. Subsequent
stable transfection experiments were performed using either 0.3 or
0.6 .mu.g/mlblasticidin.
Initial Characterization of Babesia bovis Infected Erythrocytes
After Transfection:
[0141] Plasmid pgfp-bsd-ef was designed with ef-1.alpha.
.quadrature. orf insertion sequences at the 5' and 3' regions to
target integration of the gfp-bsd cassette into the of-1.alpha.
locus. The construct was introduced into B. bovis Mo7 infected
erythrocytes by electroporation as either circular plasmid, or as
gel-purified NotI linearized plasmid in the presence or absence of
the restriction enzyme NotI in the electroporation cuvette (Table
1) (Black et al. 1995 supra). Circular pBS plasmid was used in
control cultures. Blasticidin selection was initiated using either
0.3 or 0.6 .mu.g/ml of .quadrature. blasticidin starting 24 hours
after electroporation. Parasite counts were performed daily and the
percentage of parasitized erythrocytes calculated.
[0142] Blasticidin resistant parasites emerged as early as five
days after electroporation under selection with 0.3 or 0.6 .mu.g/ml
of blasticidin (Table 1). No blasticidin resistant parasite lines
emerged in cultures containing parasites electroporated with the
control pBS plasmid (Table 1). Blasticidin resistant B. bovis
parasites were present in cultures established after
electroporation in the presence or absence of NotI using either
circular or linearized plasmids. Due to their early emergence and
rapid rate of growth in otherwise inhibitory concentrations of
blasticidin, cultures derived after electroporation with circular
plasmid in the absence of NotI (experiments 1 and 2, Table 1) were
further studied.
[0143] To select for highly blasticidin resistant lines, parasites
were cultured in either 0.64 .mu.g.quadrature.ml or 2.0 .mu.g/ml of
blasticidin. A parasite line designated 1-2-124 was able to grow in
a blasticidin concentration of 2.0 .mu.g/ml, reaching almost 4.0%
ppe two days after splitting of the cultures.
[0144] The parasite line 1-2-124 was further maintained in culture
under blasticidin selection and subjected to 1:10 splitting every
four days. Transfected parasites emitted high levels of gfp
fluorescence while fluorescence was never detectable in control
transfected parasites. Total DNA was extracted from this parasite
line 21 days after the start of the blasticidin selection, and
analyzed by PCR using primers that amplify the full size gfp-bsd
fusion orf. A .about.1.1 kb band, compatible with the size of the
gfp-bsd fusion orf, was obtained after amplification of the
transfected 1-2-124 parasite line DNA (data not shown). Sequencing
of this amplicon demonstrated 100% sequence identity with the
gfp-bsd fusion orf present in plasmid pTracer (Invitrogen). To
initially determine whether plasmid was still present in 1-2-124, 1
.mu.g of this DNA was used for plasmid rescue experiments by
transforming competent E. coli cells after electroporation. No
bacterial colonies were obtained after transformation and
ampicillin selection of E. coli competent cells at one month
following transfection, suggesting that the gfp-bsd amplicon was
not amplified from pgfp-bsd-ef or other plasmid DNA. Plasmid
indistinguishable from pgfp-bsd-ef was recoverable from culture of
another line at the same time point. These results suggested that
the transfection plasmid was integrated into the chromosome of
parasite line 1-2-124 and that gfp-bsd was consistently expressed
in the presence of blasticidin. Thus line 1-2-124 was characterized
more extensively.
Analysis of Parasite Line 1-2-124
[0145] Line 1-2-124 parasites growing either in the presence or the
absence of blasticidin selection were able to consistently express
a fluorescent product over a period of 9 months after
electroporation (at which time routine fluorescent microscopy
analysis was discontinued). To confirm expression from the fusion
gene the production of a gfp-bsd transcript and gfp-bsd fusion
protein were examined. A gfp-bsd transcript was consistently
present in transfected B. bovis after RT-PCR analysis using primers
that amplify approximately 1,200 bp of the orf . Expression of a
gfp fusion protein was demonstrated by Western blot analysis of
parasite line 1-2-124 using monospecific polyclonal antibody
against gfp. Rabbit anti-gfp antibodies bound a protein of
.about.46 kD, compatible with the expected size of the gfp-bsd
fusion protein. Anti-gfp antibody did not react with any protein in
wild type Mo7 strain parasites. An additional band of approximately
70 kD was present in immunoblots of parasite line 1-2-124 but not
in control Mo7 parasites. The identity of this protein is unknown.
However, its presence only in transfected parasites suggests that
it is either a dimer of gfp-bsd, or a second fusion protein
originating from an unexpected gfp integration event.
[0146] Growth characteristics of the blasticidin selected parasite
line 1-2-124 were examined and compared with mock pBS-transfected
control parasites in the presence or absence of 6.4
.mu.g/mlblasticidin. The results indicate that parasite line
1-2-124 is able to grow at similar rates with or without addition
of blasticidin at a concentration 10 times higher (6.4 .mu.g/ml)
than used for original selection, while control parasites
transfected with pBS were not able to grow in the presence of
blasticidin at 6.4 .mu.g/ml. In addition, no differences were
observed between growth rates of parasite cell line 1-2-124 and
control parasites in the absence of blasticidin.
The gfp-bsd Gene is Integrated into the ef-1.alpha. Locus of
Parasite Cell Line 1-2-124:
[0147] Overall, the results suggest that a gfp-bsd gene is
integrated into the genome of the B. bovis transfected line
1-2-124. Southern blot analysis using ef-1.alpha. and gfp-bsd
specific dig-labeled probes was used to confirm this, and to
determine the location of the gfp bsd gene in the B. bovis genome.
Parasite DNA was digested with the restriction enzyme BglII, which
cuts twice outside the ef-1.alpha. locus of the B. bovis genome,
but not within the transfection cassette. This digest should
generate a fragment of 12,431 bp containing the ef-1.alpha. locus
in wild type parasites. In control Mo7 strain blots, a genomic
restriction fragment of the predicted size hybridizes with the
ef-1.alpha. probe, but not with the gfp-bsd probe. There is an
upward shift of the main hybridization band in the parasite line
1-2-124 using the ef-1.alpha. specific probe, consistent with an
expected increase in the size of the BglII fragment containing the
ef-1.alpha. locus in the transfected cell line. The gfp-bsd probe
hybridized with a restriction fragment of the same size as the
ef-1.alpha. probe, but only in the transfected cell line. Southern
blot analysis showed no evidence of episomal DNA containing the
gfp-bsd gene. The lack of episomal plasmids and the upward shift of
the ef-1.alpha. restriction fragment that hybridizes with both
ef-1.alpha. and gfp-bsd probes strongly suggests that the exogenous
gfp-bsd gene is inserted into the targeted ef-1.alpha. gene
locus.
[0148] To further confirm and localize integration of the gfp-bsd
gene into the ef-1.alpha. locus, amplicons were generated by PCR
using the forward primer ef-rev-6 targeted to sequence unique to
the intergenic region of the ef-1.alpha. locus in wild type
parasites, but not present in the transfection plasmid, and the
reverse primer efrev-5 representing sequences in the ef-1.alpha.
promoter of the gfp-bsd gene of the transfection vector
pbsd-gfp-ef. The two primers used in this amplification are
separated by .about.600 bp in the genome of the wild type Mo7
strain, but are oriented in the same strand in B. bovis and thus
are unable to generate any amplicon from wild type parasites.
Amplification of genomic DNA obtained from the transfected line
1-2-124 with this set of primers resulted in a 1.5 kb band, which
was cloned into 2.1 topo vector and fully sequenced. No PCR product
was obtained either from the plasmid pgfp-bsd-ef or from wild type
Mo7 genomic DNA using this set of primers. Analysis of the 1.5 kb
PCR product indicated that it contains sequence from the genome
that is not present in the plasmid and sequence unique to the
plasmid that includes plasmid associated restriction sites. The
absence of the XhoI restriction site present in plasmid pgfp-bsd-ef
suggests that it originated from the genomic version of the gene.
In contrast, the 3' end of the r5-r6 amplicon includes the sequence
of primer xho-ef-orf-r1 exactly as it is present in the
transfection construct. The results indicate that the 1.5 kb
amplicon contains a chimera generated as a product of homologous
recombination between the genome and the transfection plasmid.
[0149] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
Sequence CWU 1
1
11133DNAArtificial SequencePrimer 1ctgacgctcg agatgccgaa ggagaagact
cac 33233DNAArtificial SequencePrimer 2cagctgctcg agatctgatc
aagggcctcg acc 33334DNAArtificial SequencePrimer 3cgtcgtgaat
tcatggcctc caaaggagaa gaac 34434DNAArtificial SequencePrimer
4taatgtgaat tcgccctccc acacataacc agag 34512DNAArtificial
SequencePrimer 5gcggccgcgg cc 12622DNAArtificial SequencePrimer
6atggcctcca aaggagaaga ac 22722DNAArtificial SequencePrimer
7gccctcccac acataaccag ag 22834DNAArtificial SequencePrimer
8catatcaagc ttctttaacg ggatgacata tatg 34935DNAArtificial
SequencePrimer 9gaccataagc ttagtaaacg atagaacaga ctaag
351035DNAArtificial SequencePrimer 10gcatcgggat ccggaacccc
caaagaggcc cgttg 351132DNAArtificial SequencePrimer 11ctagcatcct
cttagcagcc ttttgggcag ac 32
* * * * *