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Identification, characterization, and molecular cloning of the immunodominant Babesia bovis merozoite surface protein Bv42

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Title:
Identification, characterization, and molecular cloning of the immunodominant Babesia bovis merozoite surface protein Bv42
Creator:
Hines, Stephen Alan, 1955-
Publication Date:
Language:
English
Physical Description:
vii, 86 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Antigens ( jstor )
Babesiosis ( jstor )
Cattle ( jstor )
Erythrocytes ( jstor )
Membrane proteins ( jstor )
Merozoites ( jstor )
Monoclonal antibodies ( jstor )
Parasites ( jstor )
Vaccinations ( jstor )
Babesia -- genetics ( mesh )
Cloning, Molecular ( mesh )
Dissertations, Academic -- Pathology -- UF ( mesh )
Membrane Proteins ( mesh )
Pathology thesis Ph.D ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1989.
Bibliography:
Bibliography: leaves 74-84.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Stephen A. Hines.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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001140444 ( ALEPH )
22253070 ( OCLC )
AFN9788 ( NOTIS )

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IDENTIFICATION, CHARACTERIZATION, AND MOLECULAR CLONING OF
THE IMMUNODOMINANT BABESIA BOVIS MEROZOITE SURFACE PROTEIN
Bv42













By


STEPHEN A. HINES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1989




IDENTIFICATION, CHARACTERIZATION, AND MOLECULAR CLONING OF
THE IMMUNODOMINANT BABESIA BOVIS MEROZOITE SURFACE PROTEIN
Bv42
By
STEPHEN A. HINES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989


ACKNOWLEDGEMENTS
I want to thank some of the many people who contributed
to this work and helped to make my time at the University of
Florida memorable. First, it was a unique opportunity to
have had both Guy Palmer and Terry McElwain as major
professors. I hope that Guy and Terry know my gratitude,
respect, and true affection. The only problem in working
with people of their caliber is that you're left with a high
standard to measure up to. Although the other members of my
advisory committee (Tony Barbet, Lindsey Hutt-Fletcher, and
Ward Wakeland) were not as closely involved day-to-day with
my research, each of them has significantly influenced my
development as a scientist and I thank them sincerely.
I also thank my friends Drs. Richard and Catherine
Crandall who supported me when I first entered graduate
school and encouraged an interest in parasite immunology.
Likewise, Dr. Mike Burridge, Chairman of the Department of
Infectious Diseases in the College of Veterinary Medicine,
has done much to ensure the success of my work and the
project I was involved with.
ii


The monoclonal antibodies and monospecific rabbit
anti-serum against Bv42 were produced and originally
characterized at Washington State University. My thanks
goes to Travis McGuire, Lance Perryman, William Davis, Will
Goff, and David Reducker who in collaboration with my major
professors developed and provided these valuable reagents.
During my tenure as a graduate student, I have worked
alongside some remarkable people. In addition to Terry and
Guy, these were the people who made work fun and helped me
look forward to each new day. Pam Kaylor, Dr. Jennifer
Garber, and Raina DeVenere were technicians who facilitated
my work and became good friends. Stephen Kania and Vishnu
Mishra shared lab space, an interest in infectious diseases,
mutual concern for each other, and some memorable laughs.
Kelly Sloan, Lili Sotomayor, Jane Farrell, and Teri Harty
were our veterinary student "elves" who helped so much with
the animal and Babesia culture work. These four vet
students have been treasures; they reminded us of why we
were interested in veterinary medicine and brought sunshine
into the lab every summer.
Lastly, I want to thank my wife Melissa Trogdon Hines,
D.V.M., Ph.D., without whom nothing else would matter very
much.
iii


TABLE OF CONTENTS
page
ACKNOWLE DGEMENTS i i
KEY TO ABBREVIATIONS V
ABSTRACT vi
CHAPTERS
1. LITERATURE REVIEW AND RESEARCH STRATEGY
Literature Review
Introduction and Significance 1
Background Life Cycle and Disease 3
Current Methods of Control 7
Experimental Nonviable and Semidefined
Immunogens 12
Recombinant Babesia bovis Antigens 19
Summary 21
Research Strategy 23
2. MOLECULAR CHARACTERIZATION OF Babesia bovis
MEROZOITE SURFACE PROTEINS BEARING EPITOPES
IMMUNODOMINANT IN PROTECTED CATTLE
Summary 27
Introduction 28
Materials and Methods 29
Results 34
Discussion 38
3. MOLECULAR CLONING OF THE GENE ENCODING THE
IMMUNODOMINANT Babesia bovis MEROZOITE SURFACE
PROTEIN Bv42 AND ITS EXPRESSION IN Eschericia coli
Summary 50
Introduction 51
Materials and Methods 52
Results 57
Discussion 60
4. SUMMARY / CONCLUSIONS 70
REFERENCES 74
BIOGRAPHICAL SKETCH 85
iv


KEY TO ABBREVIATIONS
PCV, packed cell volume
IFA, (indirect) immunofluorescence assay
MASP, microaerophilous stationary phase
(Babesia bovis cultures)
IV, intravenous
iRBC, infected erythrocytes
PBS, phosphate buffered saline
NP-40, Nonidet P-40
PMSF, phenylmethyl-sulfonylfluoride
nRBC, normal (uninfected) erythrocytes
VBS, veronal buffered saline
SDS-PAGE, sodium dodecyl sulfate polyacrylamide
gel electrophoresis
kDa, kilodalton
kb, kilobase
v


Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IDENTIFICATION, CHARACTERIZATION, AND MOLECULAR CLONING OF
THE IMMUNODOMINANT BABESIA BOVIS MEROZOITE SURFACE PROTEIN
Bv42
By
Stephen A. Hines
December 1989
Chairman: Terry F. McElwain
Major Department: Department of Pathology and Laboratory
Medicine, College of Medicine
Eight surface-radioiodinated merozoite proteins from a
cloned, pathogenic isolate of Babesia bovis can be
immunoprecipitated by antibody from cattle that are
completely protected against clinical babesiosis. Among
these eight surface proteins, the 55 and 42 kDa molecules
are biosynthetically labeled with 3H-glucosamine. The 42
kDa glycoprotein can also be labeled with 3H-myristic acid
and partitions exclusively into the detergent phase in
Triton X-114 extracts, indicating that it is an integral
membrane protein and suggesting that it is anchored by a
glycosylphosphatidylinositol moiety. Antibody mediated
protection against B. bovis merozoites most likely requires
a high level of circulating antibody to ensure antibody-
merozoite binding during the parasite's brief
extraerythrocytic phase. Antibodies in diluted sera
vi


selectively recognize the 120, 85, 55, and 42 kDa surface
proteins. Only the 42 kDa integral membrane protein is
reactive with serum antibodies diluted > 1:16,000. Thus, we
hypothesize these immunodominant proteins, especially the
transmembrane 42 kDa glycoprotein, are important to the
induction of the protective immune response and are
candidates for an improved vaccine against babesiosis.
A 0.9 kb cDNA fragment encoding the 42 kDa
immunodominant merozoite surface antigen Bv42 was cloned
into the unique Eco RI site of the bacteriophage vector
Lambda ZAP II. The 45 kDa recombinant protein expressed in
Escherichia coli accounted for greater than 95% of the
native 42 kDa glycoprotein, including the epitope(s) defined
by monoclonal antibodies that react with the surface of live
merozoites. Antibodies prepared against recombinant Bv42
immunoprecipitated the native 42 kDa glycoprotein from
surface labeled and metabolically labeled B. bovis.
Further, antibodies against the recombinant protein bound to
and removed the 42 kDa protein recognized by surface
reactive monoclonal antibody Babb 35A4 from a preparation of
native, 35S-methionine labeled parasite antigen. The
cloning of the gene for Bv42 will now allow us to test the
ability of an immune response against this specific
merozoite surface antigen to protect cattle against
babesiosis.
vii


CHAPTER 1
LITERATURE REVIEW AND RESEARCH STRATEGY
Literature Review
Introduction and Significance
The arthropod-borne, hemoparasitic diseases of domestic
animals are significant obstacles to improved meat, milk,
and fiber production in the lesser developed tropical
nations, and represent a primary barrier to the importation
of more productive breeds of food animals [1]. Babesiosis
is a protozoal disease that is of critical importance to
cattle living in or bound for the tropical and subtropical
regions of the world. The two most important species,
Babesia bovis and B. bigemina, are estimated to endanger 500
million cattle between the thirty-second parallel north and
fortieth parallel south of the equator, where their
geographic distributions correspond to that of their vectors
[2,3].
Economic loss in babesiosis is attributable to high
mortality, which is in excess of fifty percent for
susceptible Bos taurus cattle, and to diminished production
of milk and meat in animals recovering from acute infection
and abortion. Additional losses include the high cost of
1


2
quarantine and other measures to control the spread of the
disease, as well as the costs associated with loss of market
and inability to import high yielding Bos taurus breeds [2].
In Australia, tick-borne disease costs the government and
cattle producers an estimated 42 million dollars
(Australian) per year [2]. Economic losses are also
significant in many Latin American countries, but it is
difficult to determine the economic cost of babesiosis in
much of the developing world due to lack of epidemiological
data [3].
In the U.S., eradication of Boophilus ticks and
babesiosis is estimated to save the country 500 million
dollars per year, but the potential for re-introduction of
Babesia-infected ticks from Mexico, the Caribbean, and the
Southern Hemisphere via illegal livestock importation
remains an ominous possibility [2]. The potential for
catastrophic outbreak even in areas where tick-borne disease
appears to be controlled was illustrated by the epizootics
that occurred during the Zimbabwean pre-independence war in
the mid-1970's. Nearly one million cattle died of
hemoparasitic disorders following the disruption of short-
interval dipping used to control cattle ticks [4],
In Zimbabwe and many of the endemic areas, B. bovis. B.
bigemina and the rickettsia Anaplasma margnale can share


3
the same tick vector and have vectors with shared geographic
ranges. Infections often occur in combination, resulting in
synergistic pathogenicity and a disease complex collectively
called "tick fever." Control of this complex, including
attempts to control by vaccination, will ultimately require
a strategy directed against all three organisms.
Our long-range goal is an antigenically-defined subunit
vaccine against Babesia bovis. The protection afforded by
recovery from primary infection indicates that a vaccine is
an achievable goal. Earlier work with killed, semi-defined
antigen preparations illustrates the potential for a subunit
vaccine that could induce a protective immune response in
susceptible cattle.
BackgroundLife Cycle and Disease
Transmission of bovine babesiosis is by a variety of
ticks, of which Boophilus species are most notable.
Susceptible ticks become infected when they acquire a blood
meal from an infected cow and can transmit the disease to
other cattle only after the completion of a developmental
cycle that begins in the tick midgut [5]. Here the majority
of parasites in the imbibed blood die while a small
percentage of unique intracellular forms, the putative


4
gametocytes, begin a metamorphosis. Although fusion or
fertilization of gametes has not yet been observed, the
morphologic changes that occur suggest a sexual process
[6,7], The result is a spherical body that is transformed
into an elongate ookinete. Invasion of gut epithelium is
followed by asexual division and the production of
sporokinetes (sporogony). These motile kinetes are released
by gut epithelium and disseminated via hemolymph to enter
salivary gland cells, muscle fibers, haemocytes, ovarian
cells and oocytes. Further asexual division in these cells
produces more kinetes which remain infective only for the
tick cells. Dormancy of the parasite in tick oocytes allows
for transovarial transmission to subsequent generations.
When progeny ticks mature and begin to feed, sporogony again
occurs in a manner similar to that following primary
infection in the adult. It is the invasion of salivary
gland cells by sporokinetes that initiates the form of
asexual reproduction that produces sporozoites which are
infective for cattle. Differentiation to the sporozoite
requires a feeding or temperature stimulus and the parasite
is then transmitted via saliva during tick feeding.
Following inoculation into cattle, sporozoites of
Babesia directly bind and enter erythrocytes. The


5
sporozoite becomes an intracellular feeding stage and
subsequently undergoes binary fission (merogony) to form two
to four daughter cells (merozoites). Merozoites exit the
infected erythrocyte causing host cell destruction and are
immediately infective for other erythrocytes, thereby
perpetuating the cycle of red blood cell invasion and
destruction.
The replicating population of Babesia in a host is
heterogenous due to the presence of subpopulations that
differ in antigenicity, transmissibility and virulence.
Likewise, there is evidence for antigenic differences
between isolates and for selection of subpopulations by
passage in both invertebrate and mammalian hosts [8,9].
Clinically, bovine babesiosis tends to be acute and
life-threatening, especially in Bos taurus cattle which are
more susceptible to infection than Bos indicus [10,11],
Hemolysis due to intracellular parasite multiplication and
subsequent erythrocyte destruction peaks at 7 to 20 days
post-infection. Accompanying signs include anemia, fever,
icterus, roughened hair coat, dehydration, and depression.
Pregnant animals may abort and hemoglobinuria is common.
B. bovis-infected erythrocytes sequester in visceral
vessels, notably cerebral and renal capillaries. Peripheral


6
blood parasitemias may remain below 1 percent, while
parasite rates in the cerebral capillaries typically exceed
90 percent [10]. Neurologic signs are attributed to the
blockage of cerebral capillaries by parasitized erythrocytes
and to anemic hypoxia. Also with B. bovis infections,
significant vascular changes occur when total parasite
numbers are still low and large scale hemolysis has not yet
developed [11]. Parasite-produced proteolytic enzymes are
released into the blood stream during the exit of merozoites
from infected red cells and activate certain plasma
proteins, including kallikrein and components of the
complement and clotting cascades [10]. The result is
activation of potent vasoactive mediators and a profound
hypotensive shock.
Cattle that survive the acute phase of babesiosis
typically become carriers whose blood is directly infective
to non-immune animals and tick vectors [11]. Although the
parasite is maintained in the cattle and tick populations
via these asymptomatic carriers, persistently infected
animals are also resistant to clinical disease if they are
challenged a state known as premunitv or "co-infectious
immunity" [10]. Prolonged sterile immunity can be seen in
cattle that eliminate the organism either spontaneously or
due to antibiotic treatment [12,13], This indicates that


7
living parasites need not be present in the blood stream for
animals to be resistant to disease [10]. The protection
engendered by infection is strong evidence that an effective
vaccine is an achievable goal.
Current Methods of Control
The United States' eradication of Babesia via its tick
vector has proven to be an isolated experience not likely to
be repeated on a large scale [2]. Current strategies to
control babesiosis in endemic areas are a combination of
vector reduction and immunoprophylaxis.
Vector Reduction Tick control by interval dipping and
exclusion of vectors from areas of animal congregation
remains an important mainstay in the prevention of
hemoparasitic diseases [3]. In many developing tropical
countries, acaricide programs consume a sizable percentage
of the national veterinary services budget [14]. These
programs also have important health considerations for
people involved in dipping, but they can effectively
decrease losses due to arthropod-borne diseases. The
paradox is that reduction of the tick population in endemic
areas not only retards the rate of Babesia transmission, it
also increases the number of susceptible cattle [4]. This
occurs in part because calves have a relatively high


8
resistance to clinical babesiosis for several months after
birth. Resistance is associated with poorly defined host
factors and with passive transfer of colostral antibodies
from immune dams [15]. Infection during this period
typically results in mild or inapparent disease and the
prolonged protection afforded by subclinical infection
(premunition) [15]. Effective methods of tick control
decrease the frequency of calfhood infection and therefore
also reduce the number of cattle protected by natural
infection. Subsequent changes in tick control practices in
endemic regions can upset the balance between numbers of
susceptible cattle and infected ticks, and result in
disastrous epizootics [4].
Immunoprophylaxis Vaccination is the logical method to
protect individual cattle and prevent outbreaks, especially
in highly susceptible animals that originate in non-endemic
areas or areas where Babesia infection rates are limited by
effective vector reduction programs. An effective
immunization program would increase the number of resistant
animals in an endemic area, thereby decreasing the required
dipping frequency and its associated economic burden while
restoring the enzootic stability needed to withstand
possible breakdowns in the control system [15].


9
Currently practiced methods of immunoprophylaxis use
premunization whereby resistance to the disease is
associated with the presence (at least transiently) of the
parasite in the bloodstream. Premunition is achieved by (I)
deliberate infection with an attenuated (non-pathogenic)
strain, or (II) drug cure of an infection after a
significant parasitemia has developed.
In Australia, the regional government in Queensland
annually distributes nearly one million doses of an
attenuated, live B. bovis "vaccine" [16]. The vaccine is
produced by multiple (20-30) syringe passages through
splenectomized calves and is administered as a subcutaneous
dose of infected erythrocytes [17,18]. The ensuing immune
response in vaccinates does not prevent multiplication of
challenge organisms (1O0) in erythrocytes or some early
deviations in packed cell volume or rectal temperature after
experimental challenge. However, these changes are reversed
by about the sixth day post-infection and most animals
recover without developing overt clinical signs [19].
Protection is similar to that conferred by recovery from
infection with a virulent strain transmitted by infected
ticks or direct inoculation of infected blood. Like natural
infection, protection persists for at least four years and
is effective against virulent heterologous parasites [19].



10
The Australia vaccine also has a number of serious
disadvantages [20], These include the following:
1) infected blood has a shelf life of only six days;
2) variable attenuation results in clinical disease in a
minority of recipient cattle; 3) the vaccine is a blood
product raised in calves and can serve to transmit a number
of infectious agents including other hemoparasites and
Bovine Leukosis Virus; 4) the large mass of host components
in the inoculum can sensitize the vaccinates to bovine
erythrocytic proteins and predispose to neonatal
isoerythrolysis in progeny calves; 5) passage through non-
splenectomized cattle (such as the vaccinates) can result in
a reversion to virulence thereby maintaining virulent
parasites in the population for transmission to susceptible
individuals; 6) production and distribution requires a large
centralized facility and a cold-chain; and 7) the product is
not suitable for Babesia-free regions, where its use could
result in introduction of the disease.
More recently, investigators have reported that cattle
were protected against babesiosis after infection with an
attenuated strain of B. bovis produced In vitro [21,22], In
previously unexposed cattle, the tissue culture-adapted
strains produced mild clinical signs and moderate changes in
erythrocyte packed cell volume. Production of a vaccine


11
strain of Babesia in culture would improve quality control
and decrease the need for splenectomized calves but would
not overcome many of the limitations inherent to
premunization using infected blood. Furthermore,
cultivation of hemoparasites on a large scale would be
difficult.
In Israel and elsewhere, the shelf life of an
attenuated inoculum has been extended by storage in liquid
nitrogen but the method is unwieldy [23]. Other problems
with a blood-derived, living product remain, and the low
survival rate of B. bovis after freeze/thawing necessitates
that large number of parasites be present in each dose -
thereby increasing both the mass of bovine protein
inoculated and the cost per vaccinate.
The second method of premunization involves infection
of cattle with attenuated or virulent strains of Babesia
followed by drug cure, often using the chemoprophylactic
imidocarb [24-26]. This technique is labor intensive and
requires skilled professionals to treat cattle only after a
protective immune response has had time to form but before
acute, life-threatening disease has occurred. A sterilizing
treatment administered early in the course of the infection
produces a less protective immune response than if the
treatment is given after a high parasitemia [12,24]. In


addition, chemotherapeutic drugs against Babesia are both
expensive and toxic.
12
Clearly, current methods to immunize cattle against
babesiosis are seriously flawed, but the solid protection
produced by these techniques is encouraging.
Experimental Nonviable and Semidefined Immunogens
Recent work on bovine babesiosis has focused on using
killed or semi-defined antigens to induce protective
immunity. These antigens can be classified as 1) killed
whole organism vaccines derived from infected blood,
2) antigens purified from parasitized erythrocytes by
affinity chromatography, and 3) soluble antigens from
continuous MASP cultures of B. bovis. Immunization with
such preparations does not prevent infection but can
significantly reduce morbidity and mortality. None of these
products is likely to become a satisfactory vaccine, but the
work clearly demonstrates the potential for a subunit
approach.
Whole organism vaccines
A freeze-dried suspension of killed Babesia bovis has
been shown to partially protect cattle from intravenous
challenge with 106 or 107 infected erythrocytes [27]. The


13
suspension was produced by lysis of infected cells in
distilled water and recovery of parasites by centrifugation.
Protection was manifested by fewer clinical signs and
decreased erythrocyte destruction. Subsequently, a crude
babesial antigen called infected erythrocyte antigen (IEA)
was prepared by disruption of concentrated, parasitized
erythrocytes in a French pressure cell [28]. Subcutaneous
inoculation of this product in Freund's complete adjuvant
was shown to protect Bos taurus cattle (n=4) against a
heterologous strain as effectively as recovery from a tick-
transmitted infection 12 months previously. Protection was
less effective when the challenge dose was increased from
106 to 108 infected erythrocytes. These studies indicated
that at least partial protection could be conferred with
killed antigen and suggested that antigenic variants were
not a formidable barrier to immunization. However, the
experimental preparations tested were also contaminated with
host erythrocyte antigens leading to the development of
isoantibodies [28],
Considerable effort has been directed toward
fractionation of crude babesial antigen and demonstration of
a protective immune response derived from a semidefined or
purified component [29-36]. At least two subfractions of
infected erythrocytes have been shown to induce a protective


14
immune response that is as effective as that produced by
crude antigenic material [30]. The persistent problem of
eliminating host protein from vaccine preparations has been
complicated by the discovery that some babesial antigens
complex with host protein.
Affinity purified immunogens
Three B. bovis antigens with molecular weights of 1300,
180, and 44 kDa were purified from infected blood using
monoclonal antibody affinity chromatography [37]. These
antigens could be specifically localized to the parasite or
infected erythrocyte by immunofluorescent staining. When
splenectomized calves were immunized twice with the
respective antigens in Freund's adjuvant, only the 44 kDa
antigen conferred protective immunity against a homologous
intravenous challenge of 103 organisms. Subsequently the
same 44 kDa-reactive monoclonal antibody was used to isolate
a 29 kDa antigen that had been further purified by gradient
gel electrophoresis [38], Two subcutaneous injections of
this antigen in FCA induced a protective response in nine
nonsplenectomized adult cattle that were challenged with 10*
infected erythrocytes of the homologous, virulent strain.
Whereas none of the vaccinated animals showed clinical


15
signs, three of the five unvaccinated controls were severely
affected. No animals in either group died but the
vaccinated cattle had significantly less change in PCV, body
temperature, and parasitemia.
Based on the relative protection of splenectomized
calves, the authors judged the affinity-purified 29 kDa
antigen to be as protective as crude antigen. The results
in intact adult cattle were much harder to interpret
considering variations in challenge dose, quantity of
immunogen and strains of B. bovis used for antigen
extraction and challenge. The monoclonal antibody used to
isolate the 29 kDa antigen is a labile IgM that cannot be
separated from contaminating ascites proteins without
severely affecting its biological properties. The resulting
presence of haptoglobin in the immunosorbent allows for
binding of bovine hemoglobin and subsequent elution with the
babesial antigen [38]. Native 29 kDa antigen is also
apparently complexed to large host molecules that may act as
carriers and even alter immunogenicity. Production of
sufficient purified antigen clearly remains a significant
problem in testing experimental vaccines. Nevertheless,
this work demonstrates the potential of a defined antigen
approach to develop effective immunoprophylaxis in bovine
babesiosis.


16
MASP culture derived antigens
Candidate protective antigens of Babesia bovis have
also been studied as a by-product of recent advances in in
vitro cultivation by the xnicroaerophilous, stationary phase
(MASP) technique [39,40]. Immunoelectrophoresis of culture
supernatants demonstrated at least three soluble antigens
with molecular weights in the range of 37-40 kDa [41,42],
These may be identical to three antigens that were
previously identified in the lytic extracts of infected
erythrocytes [41,29]. When monospecific rabbit antibodies
against the purified culture-derived antigens were used in
an immunofluorescence assay (IFA), two of the antigens (1
and 2) could be localized to the erythrocyte membrane or
stroma while the third was directly associated with the
parasite [43], It is this latter antigen, designated
"Antigen 3", which is postulated to represent merozoite
surface coat material that is shed into the culture media
when the merozoite invades an erythrocyte. Bovine antisera
to B. bovis culture supernatant reacts with the merozoite
surface and causes morphologic alterations in the surface
coat, aggregation, and lysis of merozoites [40], By analogy
with the merozoites of Plasmodium and species of Babesia
that infect rodents, this antibody should prevent parasite
invasion of erythrocytes and mediate their immune


17
destruction by the mononuclear phagocyte system. The other
two antigens are apparently precipitated as fibrinogen-
associated complexes that in vivo may affect coagulation,
fibrinolysis, and immune recognition of parasite proteins.
Immunization with soluble antigens in supernatants of
MASP cultures has produced conflicting results. Partial
protection was induced in four 18-month-old Bos taurus
cattle that received two subcutaneous doses (in saponin
adjuvant) and were challenged three months later with 1000
B. bovis-infected Boophilus ticks [40], Vaccinated cattle
were clinically less severely affected than identically
challenged controls.
A subsequent study showed protection of 18 yearling
heifers for four to six months after a similar vaccine
regimen and homologous challenge by intramuscular injection
of 108 infected erythrocytes [44]. Immunization induced
marked IFA serum titers that peaked 1 week after the second
vaccination and were allowed to decline to approximately
undetectable levels prior to challenge. Following
challenge, vaccinated animals developed significantly higher
IFA titers than non-vaccinated groups, and more than 50
percent of the controls died of acute babesiosis.
In both of these trials, Babesia bovis parasitemias
were observed in all challenged cattle, but in the tick


18
challenge experiment lower parasitemias were generally
associated with survival [40]. Although the initial
replication of challenge parasites caused a reduction in PCV
in all cattle, the destruction of host erythrocytes was
significantly less in the vaccinated animals that were
challenged by intramuscular injection [44].
The conflicting data on culture-derived immunization
has been published from Australia [45,46]. This group used
an identical vaccine protocol in yearling and 2-year-old Bos
taurus cattle that were challenged at 20, 70, or 179 days by
intravenous inoculation of 108 organisms of a heterologous
virulent strain. Whereas a single dose of a live attenuated
vaccine strain of B. bovis provided strong protection,
soluble antigens derived from MASP cultures of the same
organism induced only partial protection and 6 of 12 animals
required treatment to prevent death. All members of the
unvaccinated or saponin-recipient control groups needed to
be treated. The persistent problem of contaminating host
proteins was illustrated by the detection of bovine blood
group antibodies in 4 of the 5 cattle that received two
doses of culture-derived antigen [45]. Even soluble B.
bovis antigens harvested from heterologous rabbit MASP
culture systems are initially contaminated with 8 or 9
bovine proteins and the problem is more severe for antigens


19
produced in bovine systems [43].
The ability of culture-derived exoantigens to protect
against heterologous challenge has since been examined using
Latin American isolates [47,48]. Exoantigen from a
Venezuelan strain induced a significant degree of cross
protection in cattle challenged with five different
geographic isolates, while exoantigen from a Mexico isolate
produced a low degree of cross-immunity. Vaccination did
not prevent replication of parasites but did decrease the
change in packed cell volume and increase the 6 week post
challenge weight gain.
Soluble antigen from microaerophilous, stationary phase
cultures of B. bovis is unlikely to find use as an
acceptable, effective immunoprophylactic for bovine
babesiosis. The antigen is difficult and expensive to
manufacture in significant amounts and must ultimately be
freed of bovine contaminants. Nevertheless this work again
demonstrates the potential for nonviable parasite antigen as
a protective immunogen.
Recombinant Antigens of Babesia bovis
Little progress has been made in cloning genes from
Babesia species, and there is only one report describing the
immune response to a recombinant B. bovis antigen [49]. A


20
portion of the gene encoding a 44 kDa B. bovis merozoite
surface antigen was cloned into bacteriophage Lambda gtll.
This clone was selected from a genomic expression library
screened using a panel of monoclonal antibodies against
merozoite surface antigens. Cattle immunized with the
fusion protein purified from E. coli produced antibodies
that reacted with the surface of live merozoites and
immunoprecipitated a native protein of 44 kDa.
Unfortunately, the level of antibody response among
vaccinates was highly variable. The investigators are now
looking for another adjuvant or method of antigen
presentation that will produce a more consistent antibody
titer.
In another report, a positive clone was detected in a
Lambda gtll-B. bovis cDNA library screened with sera from
cattle premunized with the Australian vaccine strain of B.
bovis [50], The cDNA insert was 235 bp long and contained
coding sequences for only 44 amino acids. Antibodies
affinity purified from bovine sera by antibody select [97]
identified a high molecular weight antigen (>220 kDa) in
immunoblots of infected erythrocytes. The same antibodies
were used in an indirect immunofluorescent assay to locate
the native molecule within the parasite in infected cells.
In this study, the recombinant protein was not tested as an


21
immunogen.
Although several additional genes of B. bovis have been
cloned, the functions of these genes and the nature of their
gene products is not known [51-53]. The genes have been
used, however, to establish that isolates of B. bovis are
composed of heterogeneous subpopulations and to identify a
highly polymorphic gene family. The authors suggested, but
provided no evidence, that seguence diversity at the
polymorphic locus, designated BabR, played a role in
antigenic variation of the parasite. It remains to be shown
that the proteins encoded by BabR or any of these other
genes are relevant to protective immunity.
Summary
There is compelling evidence that a safe, effective
vaccine against bovine babesiosis is an achievable goal.
Recovery from natural infection, premunition by attenuated
strains, and drug cure of a significant parasitemia all
provide strong, enduring protection against clinical
disease. Passive transfer of immune sera and gamma-globulin
has been used to show this protection is mediated in large
part by antibody [54,55]. Antibodies directed against
surface antigens exposed during the short extracellular
phases of hemoparasite infections have been shown to prevent


22
invasion of erythrocytes and presumably opsonize parasites
for phagocytosis [56,57]. Likewise, antibodies against new
erythrocyte antigens that are encoded by hemoparasite DNA
and expressed on the surface of infected cells mediate the
removal of intracellular stages from the circulation
[56,58].
Recently, immunization with non-viable, semi-defined
parasite antigens has been shown to induce a host protective
response that significantly reduces mortality, overt
disease, and deleterious clinicopathologic changes. The
antigen-specific nature of this kind of response is
demonstrated by the effects of bovine anti-sera raised
against the three major antigens of B. bovis in supernatants
of MASP cultures [40]. These cattle are protected against
clinical disease due to a tick-transmitted homologous
challenge, and their antisera react with merozoites to cause
aggregation, lysis, and morphologic changes in the surface
coat. The failure to exploit killed antigen as an effective
vaccine reflects the complexity of the disease and the
serious problems involved in obtaining sufficient quantities
of parasite antigen that is well characterized and pure.
Many of these problems can be overcome by the application of
current molecular biology techniques.


23
Research Strategy
The long term goal of my work is development of a safe,
effective vaccine against Babesia bovis. As a part of that
goal, the specific aims of the work detailed in this
dissertation were:
(1) Identification and characterization of Babesia bovis
merozoite surface antigens using antibodies from cattle
protected against babesiosis.
(2) Identification and characterization of cDNA clones
expressing recombinant merozoite surface antigens in
Escherichia coli.
A prerequisite for an antigenically defined vaccine
against B. bovis is the identification of parasite antigens
which are necessary or sufficient for the induction and
expression of a host-protective immune response. Toward
that end, I identified and characterized candidate host-
protective antigens located on the surface of the merozoite
of B. bovis. These antigens were radioiodinated on live
merozoites by a surface specific method (lactoperoxidase)
[59-61] and immunoprecipitated using antibodies from cattle
proven to be protected against babesiosis.


24
Kinetic models predict that merozoite surface antigens
which are relevant to protective immunity must induce high
levels of antibody to ensure antibody-merozoite binding
during the parasite's brief extracellular phase [62]. Of
eight surface proteins identified using antibodies from
protected cattle, four were shown to be immunodominant blood
stage antigens. A 42 kilodalton (kDa) glycoprotein,
designated Bv42, elicited the highest antibody titer. I
hypothesized that the immunodominant surface proteins,
especially Bv42, were important to the induction of the
protective immune response and set out to clone the gene(s)
encoding one or more of these candidate immunogens.
Polyadenylated mRNA collected from asynchronous MASP
cultures of a cloned B. bovis isolate was used to construct
a cDNA expression library in E. coli [63-66]. cDNA was
synthesized by the Gubler/Hoffman technique, which is highly
efficient for producing full length cDNA copies of isolated
mRNA [67]. cDNA was cloned into the unique Eco RI site in
the Lac Z gene of bacteriophage Lambda ZAP II (Stratagene,
La Jolla, CA, USA).
Lambda ZAP II bacteriophage was chosen as a cloning
vector for the following reasons: 1) lambda phage is highly
efficient for in vitro packaging and introduction of DNA
into a bacterial host [69], (2) Lambda ZAP II has a multiple


25
cloning site which simplifies subcloning of the insert,
especially if it contains an internal Eco RI site, (3)
addition of a helper phage to Lambda ZAP II allows in vivo
excision of a circularized phagemid which contains the
insert and can be rescued for plating onto E. coli [70],
(4) DNA inserted into the Eco RI cloning site of Lambda ZAP
II is expressed as a fusion protein containing only 3.9 kDa
of vector encoded polypeptide, and (5) the E. coli host
strain carries the lac repressor gene, lac I, so that the
potentially deleterious expression of recombinant genes can
be conditionally down regulated.
Recombinant plaques were immobilized on nitrocellulose
membranes and screened for the expression of immunodominant
merozoite surface antigens using polyclonal, monospecific
rabbit sera [71]. A clone encoding the immunodominant
merozoite surface protein Bv42 was selected for further
characterization. In order to prove definitively that the
gene expressed in E. coli encoded Bv42, antibodies were
raised against the recombinant antigen in rabbits and cattle
and shown to recognize the native molecule.
A recombinant merozoite surface protein proposed for
immunization should bear surface epitopes to induce a
protective immune response against B. bovis merozoites.
Importantly, the recombinant Bv42 protein from E. coli is


26
recognized by Babb 35A4, a monoclonal antibody against
native Bv42 that reacts with the surface of live merozoites
and thus defines a surface exposed epitope [72]. This
observation indicates that the recombinant protein closely
reproduces at least one surface exposed region of native
Bv42.


CHAPTER 2
MOLECULAR CHARACTERIZATION OF BABESIA BOVIS MEROZOITE
SURFACE PROTEINS BEARING EPITOPES IMMUNODOMINANT IN
PROTECTED CATTLE
Summary
Eight surface-radioiodinated merozoite proteins from a
cloned, pathogenic isolate of Babesia bovis can be
immunoprecipitated by antibody from cattle that are
completely protected against clinical babesiosis. Among
these eight surface proteins, the 55 and 42 kDa molecules
are biosynthetically labeled with 3H-glucosamine. The 42
kDa glycoprotein can also be labeled with 3H-myristic acid
and partitions exclusively into the detergent phase in
Triton X-114 extracts, indicating that it is an integral
membrane protein and suggesting that it is anchored by a
glycosylphosphatidylinositol moiety. Antibody mediated
protection against B. bovis merozoites most likely requires
a high level of circulating antibody to ensure antibody-
merozoite binding during the parasite's brief
extraerythrocytic phase. Antibodies in diluted sera
selectively recognize the 120, 85, 55, and 42 kDa surface
proteins. Only the 42 kDa integral membrane protein is
reactive with serum antibodies diluted > 1:16,000. Thus, I
27


28
hypothesize that these immunodominant proteins, especially
the transmembrane 42 kDa glycoprotein, are important to the
induction of the protective immune response and are
candidates for an improved vaccine against babesiosis.
Introduction
Animals that survive natural field infection or that
recover from infection with an attenuated strain are
protected against clinical disease [19,74]. These
observations indicate that the host can mount a protective
immune response after exposure to parasite antigens.
Merozoite surface proteins are important in the pathogenesis
of babesiosis due to their role in the parasite's
recognition of, attachment to and penetration of host eryth
rocytes [75], Accessible to the immune system and present
on the disease causing stage, these antigens are potential
targets of the protective immune response and, therefore,
candidates for vaccine development. The ability of
merozoite surface proteins to elicit a protective immune
response has been shown with Plasmodium falciparum. The
precursor protein for three major merozoite surface
polypeptides can induce complete protection against a lethal
challenge in immunized monkeys [76]. Importantly, post-
translational modification of two of these P. falciparum


29
proteins by glycosylation may contribute to their
antigenicity [77].
Seven B. bovis merozoite surface antigens have
previously been identified using monoclonal antibodies
against surface exposed epitopes [72,49], These monoclonal
antibodies reacted with live merozoites in indirect
immunofluoresence assays, and immunoprecipitated
radiolabeled proteins of 16, 37, 42, 44, 60, 85, and 225
kDa. In this study, additional methods are used to more
comprehensively detect merozoite surface targets and to
characterize the post-translational modification of these
proteins. Importantly, a cloned isolate of B. bovis was
examined in order to avoid confusion generated by
intraspecific antigenic variation [51,78]. Antibodies from
each of five cattle completely protected against clinical
babesiosis identify immunodominant parasite antigens that I
hypothesize are relevant to the protective immune response.
Materials and Methods
Immune animals A Mexico isolate of B. bovis was cloned in
vitro by limiting dilution as described [66]. Five 4- to 5-
month old Holstein steers were each infected by intravenous
(IV) subinoculation of 5 x 107 infected erythrocytes (iRBC)
from a splenectomized calf. The cattle were challenged


30
three times IV with 108 iRBC each, once by subinoculation
and twice using culture derived parasites. At 127 days post
inoculation, the five cattle and three weight-matched,
previously uninfected controls were challenged IV with 109
iRBC from culture. The packed cell volume and parasitemia
of all animals was monitored daily. Sera used in the
following experiments was collected at 126 days post
inoculation.
Microaeroohilous stationary phase cultures \ Metabolic
labeling The cloned isolate was cultured in 24-well plates
or flasks by a modification of a previously described method
[39], For metabolic labeling, parasites were grown for 12
hours in complete medium or methionine-deficient medium. At
12 hours the media were replaced with media containing 200
or 400 uCi 35S-methionine, 3H-glucosamine, or 3H-myristic
acid. Cultures were harvested at 24 hours and iRBC's were
washed five times in cold phosphate buffered saline (PBS)
prior to extraction in lysis buffer [50 mM Tris pH 8.0, 1%
(v/v) Nonidet P-40 (NP-40), 5mM EDTA, O.lmM N-alpha-p-tosyl-
L-lysyl-choromethylketone, and ImM phenylmethyl-
sulfonylfluoride (PMSF)]. No radiolabel was incorporated
into protein in uninfected MASP control cultures that
received radioisotope.


31
Merozoite Purification A high percentage of parasitized
erythrocytes was produced in vitro by progressively reducing
the packed cell volume [79]. Merozoites were purified by a
previously described technique [80]. Viability was assessed
by mixing merozoites with 6-carboxyfluorescein diacetate.
This method, previously used for demonstrating viability of
Babesia sp. [72,81], relies on an intact cell membrane to
prevent leakage of fluorescein from the cell [82].
Viability was expressed as the percentage of total parasites
that were fluorescent.
Surface radioiodination Merozoite surface proteins were
radioiodinated using lactoperoxidase as previously described
[83]. Normal red blood cells (nRBC) from uninfected control
cultures were collected, washed three times in PBS, and
radiolabeled identically. An equivalent number of nRBC
ghosts were prepared by lysing washed uninfected cells from
control cultures by freeze/thaw in liquid nitrogen. Ghosts
were washed free of hemoglobin in PBS by multiple
centrifugations at 35,000 X g, 20 min., 4C and discarding
the supernatant until it was clear. The final pellet was
resuspended in PBS and radiolabeled with 125I using
lactoperoxidase.


32
Immunoprecipitation Immune sera collected one day prior to
the final challenge experiment were adsorbed three times
with an equal volume of packed intact nRBC's and three times
with an equal volume of nRBC ghosts (prepared as described
above). Radiolabeled B. bovis lysate was ultracentrifuged,
sonicated, and filtered as described previously [83]. This
antigen was then incubated overnight at 4C with 15 ul of
bovine serum or 15 ul of a dilution of serum in veronal
buffered saline (VBS) pH 7.4, containing 1% (v/v) NP-40.
One hundred and fifty microliters of 10% (v/v) formalinized
Protein G-bearing Streptococcus (Omnisorb; Calbiochem; San
Diego, CA) in VBS pH 7.4, 1% (v/v) NP-40, 0.1% (w/v) gelatin
were added and incubated for 2 hours at 4C [84]. The
precipitates were washed twice with VBS, 1% (v/v) NP-40;
four times with VBS, 2 M NaCl, 1% (v/v) NP-40, 10 mM EDTA;
and twice more with VBS, 1% (v/v) NP-40. The precipitated
protein was eluted by boiling in sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
containing 2% SDS and 2.5% 2-mercaptoethanol [83].
SDS-PAGE Immunoprecipitates were electrophoresed under
reducing conditions in 7.5 to 17.5% continuous gradient
polyacrylamide gels [83,85].


33
Phase separation of Triton X-114 Washed iRBC's from 35S-
methionine labeled cultures were lysed in 10 mM Tris, 154 mM
NaCl pH 7.4, 1% (v/v) Triton X-114, ImM PMSF at 0-4C and
frozen at -20C. For protein separation, the antigen
extract was first ultracentrifuged, sonicated and filtered
as previously described [83]. The Triton X-114 solubilized
proteins (107 protein bound counts per minute in a volume of
2.0 ml) were subjected to temperature-dependent phase
separation [86]. One million protein bound counts per
minute of each phase and the starting antigen preparation
were immunoprecipitated as described above.
Immunoblotting Parasite antigen for immunoblotting was
prepared from MASP erythrocyte cultures with approximately
25% parasitized erythrocytes. Briefly, iRBC's and nRBC
controls were washed two times in cold Puck's saline G and
two times in cold PBS, resuspended in PBS, counted, and
frozen at -20C. To remove hemoglobin from lysed cells the
samples were thawed and washed in cold PBS (43,000 X g, 20
min. 4C) until the discarded supernatant was clear. The
final pellet was detergent extracted with lysis buffer and
processed as described [83]. Twenty five million iRBC's or
an equivalent total number of nRBC's (10s) were mixed with


34
3X SDS-PAGE sample buffer, boiled for 3 minutes,
electrophoresed in a 7.5 to 17.5% continuous gradient
polyacrylamide gel, and electrophoretically transferred to a
nitrocellulose membrane [87]. The nitrocellulose was
blocked for 4-6 hours in VBS (pH 7.4) containing 0.25% (v/v)
Tween-20, 0.25% (w/v) gelatin (blocking buffer), and reacted
overnight at room temperature with immune sera diluted in
blocking buffer [84]. After washing three times in blocking
buffer and two times in VBS containing 0.1% (w/v) gelatin,
the nitrocellulose was incubated for 2 hours at room
temperature with 2.5 uCi of 125I-Protein G (Amersham Corp;
Arlington Heights, IL) diluted in VBS containing 0.1% (w/v)
gelatin. The nitrocellulose was washed twice with VBS, 0.1%
(w/v) gelatin and four times with 1M NaCl, 10 mM EDTA, 0.25%
(v/v) Tween-20, and exposed to X-ray film with an
intensifying screen at -70C.
Results
Challenge of immune animals All five cattle infected and
repeatedly challenged with a cloned isolate of B. bovis were
completely protected against a homologous challenge.
Control cattle in the final challenge experiment experienced
a 28% reduction in packed cell volume (Fig. 2-1) (p<.0005
when compared to previously infected cattle; Student's


35
paired t test), while only the initial infection caused a
significant reduction in packed cell volume in the exper
imental group (39%). Despite producing extensive hemolysis,
this isolate seldom produces a circulating parasitemia
greater than 0.1% in non-splenectomized cattle. Immune sera
used in the following experiments were collected one day
prior to the final challenge.
Identification and characterization of merozoite surface
proteins Merozoites purified from culture were 95-100%
viable by 6-carboxyfluorescein diacetate staining.
Immunoprecipitation of surface radioiodinated proteins with
serum antibodies from all five protected animals identified
seven dominant surface proteins with relative molecular
weights of 250, 120, 98, 85, 55, 42, and 37 kilodaltons
(Fig. 2-2A). The 250 kDa protein does not enter the
resolving gel in a standard 14 cm. 7.5-17.5% polyacrylamide
gel but is clearly resolved in a 25 cm. gel (Fig. 2-2B). An
eighth protein of 25 kDa was immunoprecipitated by serum
antibodies from two calves (lanes 7 and 10). Control
immunoprecipitation of radioiodinated intact nRBC and nRBC
ghosts revealed no specific bands on SDS-PAGE (data not
shown).


36
To confirm that these proteins were parasite derived,
the immune sera were used to immunoprecipitate 35S-
methionine metabolically labeled parasite proteins. The
immunoprecipitated 35S antigen profile was identical in all
five protected animals. When run side by side with 35S-
methionine labeled precipitated proteins, the radioiodinated
120, 98, 85, 55 and 42 kDa surface proteins comigrate with
metabolically labeled antigens (Fig. 2-3) The 25 and 37
kDa proteins were inconsistently precipitated from 35S-
methionine labeled antigen (Fig. 2-6).
Surface protein glycosylation and myristylation were
examined by immunoprecipitation of blood stage antigen after
in vitro incorporation of 3H-glucosamine and 3H-myristic
acid. Comigration of 125I and 3H-labeled antigen in a
polyacrylamide gel shows that the 55 and 42 kDa surface
labeled proteins are glycosylated and the 42 kDa
glycoprotein is myristylated (Fig. 2-4). Neither radiolabel
was incorporated into host protein in nRBC control cultures.
The 34 kDa glycoprotein seen in lane 4 of Figure 4 cannot be
consistently surface labeled and immunoprecipitated.
Temperature dependent phase separation of Triton X-114
was used to determine whether the surface exposed antigens
were transmembrane proteins. Parasite proteins were
metabolically labeled in culture with 35S-methionine and


37
solubilized in 1% Triton X-114 at 0-4C. The antigen
preparation was wanned above the detergent's cloud point
(20C) and separated into aqueous and detergent phases by
centrifugation. Immunoprecipitation from each phase and the
starting solution shows that the 42 kDa antigen partitions
exclusively into the detergent phase (Fig. 2-5). The other
surface proteins partition into the aqueous phase.
Immunoqenicitv To determine the relative immunogenicity of
these surface proteins in protected animals, antibodies in
sequentially diluted sera were used to immunoprecipitate
metabolically labeled antigen. The reactivity of these
antibodies against the majority of 35S-labeled proteins is
lost at a 1:160 serum dilution (Fig. 2-6). However the 120,
55, and 42 kDa surface proteins remain reactive at serum
dilutions of 1:160 to 1:640. Because interpretation of
immunoprecipitation is dependent on the specific
radioactivity of labeled proteins, antibodies in diluted
sera were also examined for their reactivity with parasite
antigens by immunoblotting (Fig. 2-7). Compared to
undiluted serum, antibodies in immune serum diluted 1:500
recognize a limited number of blood stage proteins,
including proteins that migrate at the same molecular weight
as the 120, 85, 55 and 42 kDa surface antigens. A protein


38
that comigrates with the 42 kDa integral membrane
glycoprotein is consistently recognized at serum dilutions
>1:16,000.
Discussion
The immune response to Babesia bovis infection protects
surviving cattle against babesiosis. Previous work has also
shown that nonviable parasite antigens will induce partial
protection in cattle, and supports a subunit approach to
vaccine development [27,34,40,37]. My strategy for an
improved vaccine is to identify and characterize surface
antigens of the merozoite, the blood stage that is infective
for the erythrocyte and accessible to the host immune
system. Native and recombinant parasite antigens can then
be purified, or expressed by recombinant vaccine vectors,
and evaluated as immunogens.
Seven B. bovis merozoite surface proteins have been
identified on an uncloned Mexico isolate using monoclonal
antibodies [72,49]. By indirect immunofluorescence, these
antibodies reacted with the surface of live merozoites and,
therefore, defined proteins containing regions that are
accessible to antibody binding. Four proteins of 37, 42,
44, and 85 kDa were distributed over the entire surface of
the merozoite and could be surface radioiodinated.


39
In the present study, antibodies in sera from cattle
that are proven to be protected against virulent challenge
also react with surface iodinated proteins of 37, 42 and 85
kDa. The 42 and 85 kDa surface proteins can be
immunoprecipitated using the previously descibed monoclonal
antibodies (data not shown). In addition, antibodies in
these sera identify five merozoite surface proteins of 250,
120, 98, 55, and 25 kDa not previously detected with
monoclonal antibodies. The parasite specificity of all
eight surface antigens is confirmed by the failure of serum
antibodies from protected cattle to react with iodinated
normal erythrocyte proteins and by the biosynthetic labeling
of these proteins in cultures in which only parasite mRNA is
translated [83,81], The use of a cloned isolate in this
work is significant in that all eight surface exposed
proteins are expressed on the merozoite and do not represent
variants in subpopulations. Uncloned B. bovis isolates,
including the current Australia vaccine strain, are composed
of subpopulations that differ in antigenicity, virulence and
abundance within an isolate [51,78].
To induce immunity, a recombinant parasite antigen may
need to be presented by a eukaryotic expression vector that
can post-translationally modify the protein and preserve
native epitopes including carbohydrate moieties [73].


40
Carbohydrate side chains are important for the antibody
reactivity of Plasmodium falciparum antigens and are likely
to be significant in Babesia spp. [77]. By
immunoprecipitation of 3H-glucosamine labeled parasite
antigen, the 55 and 42 kDa B. bovis molecules are shown to
be glycosylated. The contribution of carbohydrate epitopes
to the immunogenicity of these proteins needs further study.
The 42 kDa merozoite surface glycoprotein was
identified as a transmembrane protein using phase separation
in Triton X-114. In this technique, integral membrane
proteins interact with the nonionic detergent via their
hydrophobic domain and when warmed above 20C can be
separated from hydrophilic proteins (which are excluded from
detergent micelles) by centrifugation [86]. The 35S-
methionine labeled 42 kDa molecule was immunoprecipitated
almost exclusively from the detergent phase. Additional
surface exposed proteins that separate into the aqueous
phase may be peripheral membrane proteins or may be
partitioning anomalously under these conditions [88,89].
Recognized causes of anomalous partitioning of integral
membrane proteins into the aqueous phase of Triton X-114
include (1) large hydrophilic moieties such as
carbohydrates, (2) the formation of oligomers which protect
hydrophobic domains, and (3) noncovalent association with


41
peripheral (hydrophilic) proteins such as those from the the
cytoskeleton (90). The strong hydrophobicity of the 42 kDa
glycoprotein may indicate that appropriate presentation,
possibly in the context of a membrane, will be required for
it to induce an effective immune response against the
merozoite.
Most integral membrane proteins are anchored in the
membrane by a hydrophobic stretch of amino acids. However,
a significant number of eukaryotic integral membrane
proteins are held in the membrane by a phosphatidylinositol
lipid moiety [91]. These proteins, which include the major
surface antigens of the parasitic protozoa Trypanosoma
brucei. Leishmania manor and Plasmodium falciparum, are
covalently modified at their C-terminus with a glucosamine-
containing carbohydrate chain and the fatty acid myristate
[92-94], By analogy with these protozoal surface proteins,
the incorporation of both 3H-glucosamine and 3H-myristic
acid into the 42 kDa protein suggests that this molecule is
embedded in the merozoite membrane by a similar glycosyl-
phosphatidylinositol anchor [95]. The previously described
T. brucei and L. major anchors are sensitive to specific
degradation with phospholipases, releasing the protein from
the cell surface and exposing a cross-reactive epitope.
Similar studies are needed to characterize the role of


42
myristic acid and glycans in anchoring the B. bovis 42 kDa
protein.
In general, antibodies that inhibit P. falciparum
merozoite invasion of erythrocytes in vitro must be present
in high concentrations [62]. High antibody levels ensure
that enough surface specific antibody will be available to
effectively react with parasites during their short
extracellular phase. Cattle that are immune to infection
with a cloned isolate of Babesia bovis produce high titered
antibody that is preferentially directed against four
merozoite surface proteins with relative molecular weights
of 42, 55, 85, and 120 kDa. The 42 kDa integral membrane
glycoprotein that elicits the highest antibody titer has
previously been shown to be species specific and to contain
epitopes conserved in at least four geographic isolates [96
and unpublished data]. The preferential response of
protected animals to these four proteins, particularly the
42 kDa glycoprotein, leads me to hypothesize that these
parasite proteins are important in the induction of
protective immunity. Native and recombinant protein are
being isolated to test this hypothesis.


43
DAYS POST-INFECTION
FIGURE 2-1 Animals previously infected with the cloned
Mexico isolate of B. bovis are immune to virulent challenge.
Percentage change in PCV was measured daily in previously
infected (), n=5, and uninfected ( ), n=3, animals
following inoculation with 109 infected erythrocytes of the
cloned isolate.


44
FIGURE 2-2 Antibodies in immune sera immunoprecipitate
eight B. bovis merozoite surface proteins. A. Gradient
SDS-PAGE (14 cm.). Radioiodinated merozoite proteins were
immunoprecipitated using Protein G-bearing Streptococcus
alone (Lane 0),_pre-infection sera (Lanes 1-5) or immune
serum (Lanes 6-10) from animals B101 (Lane 1,6), B102 (Lane
2,7), B108 (Lane 3,8), B109 (Lane 4,9), and B116 (Lane
5,10). B. A 250 kDa radioiodinated protein (arrow)
immunoprecipitated by antibodies in immune sera was more
clearly resolved in a 25 cm. gradient SDS-polyacrylamide
gel.


45
FIGURE 2-3 Surface radiolabeled B. bovis merozoite proteins
(Lanes 1,7) comigrate with metabolically labeled antigens
(Lanes 2-6). Radioiodinated merozoite surface proteins were
immunopreicipitated using immune sera form animals B102 and
B116 (Lanes 1 and 7, respectively). 35S-methionine labeled
antigens were immunoprecipitated using immune sera from B101
(Lane 2), B102 (Lane 3), B108 (Lane 4), B109 (Lane 5), and
B116 (Lane 6).


46
1 2 3 4 5
200-
92.5-
30-
FIGRE 2-4 Post-translational modification of merozoite
surface proteins. Antigen was metabolically labeled in
erythrocyte cultures with 3H- myristic acid (Lanes 1,2) or
3H-glucosamine (Lanes 3,4). Antibodies in immune serum
(animal B116) immunoprecipitated radiolabeled proteins from
infected (Lanes 1,4) but not uninfected (Lanes 2,3)
cultures. The 42 kDa myristylated B. bovis protein and the
42 and 55 kDa glycosylated proteins comigrated with
radioiodinated merozoite surface proteins (arrows) that were
immunoprecipitated using immune sera (Lane 5, B109).


47
FIGURE 2-5 The 42 kDa B. bovis merozoite surface protein
partitions into the detergent phase of Triton X-114.
Following temperature dependent phase separation, immune
serum from animal B101 was used to immunoprecipitate 35S-
methionine labeled antigen from the starting 1% Triton X-114
antigen extract (lane 1), the aqueous phase (lane 2) and the
detergent phase (lane 3). Arrow indicates the 42 kDa
protein.


48
1 2 3 4 5 6 7
FIGURE 2-6 Immune cattle produce high titered antibody that
is preferentially directed against the 125, 55, and 42 kDa
merozoite surface proteins. 35S-methionine metabolically
labeled B. bovis proteins were immunoprecipitated using
undiluted preinfection serum from animal B101 (Lane 1) and
B101 immune sera that was undiluted (Lane 2) or diluted
1:40, 1:80, 1:160, 1:320, and 1:640 (Lanes 3,4,5,6, and 7,
respectively). Arrows indicate the immunodominant 120 kDa,
55 kDa and 42 kDa proteins.


49
FIGURE 2-7 Immune cattle produce high titered antibody that
preferentially binds the 120, 55, 42, and 85 kDa merozoite
surface proteins in immunoblots. B. bovis blood stage
antigen was transblotted and probed using preinfection serum
from animal B101 diluted 1:500 (Lane 1) and B101 immune sera
diluted 1:500, 1:4000, 1:8000, and 1:16,000 (Lanes 2,3,4,
and 5,respectively). Each lane contains 2.5 x 107 iRBC.
Arrows indicate the 120, 85, 55, and 42 kDa parasite
proteins.


CHAPTER 3
MOLECULAR CLONING OF THE GENE ENCODING THE IMMUNODOMINANT
BABESIA BOVIS MEROZOITE SURFACE PROTEIN Bv42 AND ITS
EXPRESSION IN ESCHERICHIA COLI.
Summary
Bv42 is the immunodominant merozoite surface antigen of
Babesia bovis and a candidate immunogen against bovine
babesiosis. A 0.9 kb cDNA fragment encoding Bv42 was cloned
into the unique Eco RI site of the bacteriophage vector
Lambda ZAP II. The 45 kDa recombinant protein expressed in
Escherichia coli accounted for greater than 95% of the
molecular weight of the native 42 kDa glycoprotein,
including the epitope(s) defined by monoclonal antibodies
that react with the surface of live merozoites. Antibodies
prepared against recombinant Bv42 immunoprecipitated the 42
kDa glycoprotein from surface labeled and metabolically
labeled B. bovis. Further, antibodies against the
recombinant protein bound to and removed the 42 kDa protein
recognized by surface reactive monoclonal antibody from a
preparation of native, 35S-methionine labeled parasite
antigen.
50


51
Introduction
Cattle that recover from infection with an attenuated
vaccine strain of Babesia bovis are protected against
clinical disease induced by arthropod-borne sporozoite
challenge [19]. Infection with such an attenuated blood
product has serious practical limitations, including
variation in infective dose and the potential transmission
of other blood borne diseases [74], However, the observation
that an immune response to blood stages protects animals
challenged via the arthropod vector suggests strongly that a
host immune response to appropriate blood stage antigens
would be sufficient to protect cattle against natural
Babesia infections. My strategy toward the development of
an improved vaccine against Babesia bovis is to identify,
isolate, and test antigens exposed on the surface of the
merozoite. Merozoite surface proteins are accessible to the
host immune system and play a functional role in erythrocyte
invasion [75].
Surface reactive antibody that is relevant to
protection will likely be present at high levels to ensure
antibody-merozoite binding during the parasite's short
extracellular phase [65]. Using antibodies from cattle
completely protected against clinical babesiosis, I
previously identified four immunodominant merozoite surface


52
antigens having apparent molecular weights of 42, 55, 85,
and 125 kDa in SDS-polyacrylamide gels [98], Bv42 is the
surface antigen that elicits the highest antibody titer.
This 42 kDa glycoprotein has a surface exposed region
defined by monoclonal antibodies [72] and contains epitopes
conserved in isolates from Mexico, Honduras, Australia and
Argentina [96 and unpublished data], I report here the
molecular cloning and expression of the gene encoding Bv42.
Importantly, the recombinant protein expressed in
Escherichia coli (E. coli) is a faithful immunologic replica
of Bv42 that can be used to induce antibodies against the
native glycoprotein.
Materials and Methods
Parasites A cloned Mexico isolate was cultivated in long
term cultures by a previously described technique [39].
Parasite antigens were metabolically labeled in vitro with
35S-methionine or 3H-glucosamine as described [98].
Merozoites were isolated from cultures with a high
percentage of parasitized erythrocytes [80,79] and surface
radioiodinated using the lactoperoxidase technique [83,98].
Merozoite viability was greater than 95% as assessed by
retention of 6-carboxyfluoroescein diacetate [81,82].


53
B. bovis cDNA Expression Library Erythrocytes from
asynchronous B. bovis-infected blood cultures were washed
three times in Puck's saline G and stored frozen in liquid
nitrogen. Cells were thawed in lysis buffer containing 0.2
M NaCl, 0.2 M Tris-HCl pH 7.5, 1.5 mM MgCl2, 2% SDS (w/v),
and 200 ug/ml Proteinase K and then incubated in lysis
buffer at 46C for 2 hours [65]. The NaCl concentration of
the lysate was adjusted to 0.5 M and poly [A+] RNA was
isolated by batch adsorption with oligo(dT) cellulose [65].
RNA eluted with 0.01 M Tris-HCl pH 7.5 was used to prepare a
blood stage cDNA library in Lambda ZAP II (Stratagene, La
Jolla, Ca, USA) [70] by a modified Gubler and Hoffman method
using Eco RI adapters (Pharmacia LKB, Piscataway, NJ, USA)
[67]. The cloned insert in plaque purified lambda phage was
subcloned into Bluescript SK(-) phagemid using the in vivo
excision capabilities of Lambda ZAP II [70].
Immunoscreeninq Plaque lifts onto isopropyl thiogalacto-
pyranoside soaked nitrocellulose were screened using
monospecific rabbit anti-Bv42 antisera (R-914) followed by
I-Protein A and autoradiography [71,49]. Rabbit R-914 had
been immunized with native Bv42 protein immunoaffinity
purified using Babb 35A4, a previously described monoclonal


54
antibody [72]. Positive plaques were tested for reactivity
with monoclonal antibodies that recognize a Bv42 surface
exposed epitope [72,49] as well as an isotype control
monoclonal antibody and normal rabbit sera. Recombinant
phagemid excised from positive, plaque purified lambda phage
was tested for expression by a similar method using colony
lifts from transformed, ampicillin resistant E. coli (XL1-
Blue strain) [71].
Restriction Enzyme Digestion Lambda rBv42 phagemid DNA was
isolated from bacteria by anion exchange chromatography
(Qiagen Inc., Studio City, CA) and restriction enzyme
digested by standard methods [99]
Immunoblottinq E. coli host strain XLl-Blue containing the
lambda rBv42 phagemid, an unrelated recombinant Anaplasma
marginale-Bluescript phagemid, or no phagemid was grown in
liquid culture to OD600= 0.9-1.2 Bacteria washed three times
in phosphate-buffered saline (PBS) were lysed in PBS by
sonication and several cycles of freeze/thaw. The resulting
bacterial lysate was cleared by ultracentrifugation,
filtered, and sonicated again before freezing in aliquots at
-70C [83]. Antigen from equivalent numbers of bacteria or


55
from 2.5 X 107 B. bovis infected erythrocytes were separated
by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and transblotted to nitrocellulose filters
[87,98]. Nitrocellulose filters were probed as described
for immunoscreening.
Antisera against recombinant Bv42 A whole lysate of E. coli
containing lambda rBv42 phagemid was prepared as described
above. Two rabbits (R-S19, R-S20) and two 4-5 month old
steers were immunized four times with a crude PBS lysate of
whole bacteria that had been disrupted by sonication and
multiple cycles of freeze/thaw and emulsified in Freund's
adjuvant. Two additional rabbits (R-S21 and R-S22) were
immunized four times with PBS soluble bacterial antigen that
had been cleared by ultracentifugation before emulsification
in Freund's adjuvant. Control anti-E. coli antibody was
produced by immunizing rabbits with a similar crude PBS
lysate of XLl-Blue without Bluescript phagemid (R-126) or
XLl-Blue with Bluescript phagemid encoding an unrelated
Anaolasma marginale antigen (R-108).
Radioimmunoorecipitation / SDS-PAGE Radiolabeled B. bovis
antigen was processed and immunoprecipitated using Protein
A-bearing Staphylococcus aureus as previously described


56
[81,83]. The immunoprecipitates were electrophoresed in 7.5
to 17.5% gradient SDS-polyacrylamide gels and detected by
autoradiography [83].
Removal of native Bv42 from an antigen preparation bv
immunoorecipitation using antibody against rBv42
35S-methionine labeled B. bovis antigen (106 protein bound
counts per minute/tube) was incubated with 10 ul of rabbit
R-S21 antisera or 5 ug of Babb 35A4 monoclonal antibody.
Following precipitation of immune complexes with protein A-
bearing Staphylococcus aureus, the supernatant antigen from
the R-S21 tube was transferred to a new tube and incubated
with another 10 ul of R-S21 antisera. Immune complexes were
again precipitated using protein A-bearing bacteria.
Antigen that remained in the supernatant from this
immunoprecipitation was transferred to a third tube and
incubated with 5 ug of Babb 35A4. Antigen recognized by
monoclonal antibody was precipitated by sequential
incubation with rabbit anti-mouse IgG antisera and protein
A-bearing Staphylococcus aureus. All immunoprecipitates
were eluted from bacteria and examined by SDS-PAGE as
described previously [81,83].


57
Results
Identification of a cDNA clone encoding Bv42 The B.
bovis cDNA library, containing 1.2 X 106 recombinant plaque
forming units, was screened for expression using a
monospecific rabbit antiserum (R-914) against Bv42. A
positive clone, designated Lambda rBv42, was plaque purified
and shown in plaque lifts to express a protein that reacted
with Babb 35A4, a monoclonal antibody against native Bv42
that recognizes an exposed epitope on the surface of live
merozoites [72], Positive plaques did not react with normal
rabbit serum or isotype control monoclonal antibody.
Lambda rBv42 was subcloned into pBluescript SK(-)
phagemid in E. coli XLl-Blue using the in vivo excision
properties of Lambda ZAP II [70], Bacteria containing the
recombinant phagemid expressed a protein recognized by R-914
and two surface reactive monoclonal antibodies against
native Bv42 (Babb 35A4 and 23.10.36; Fig. 3-1) [6,7].
Positive bacterial colonies were not recognized by normal
rabbit serum or control monoclonal antibody, and none of the
Bv42 reactive antibodies reacted with host strain bacteria
not containing the phagemid.


58
Size and location of insert The phagemid encoding
recombinant Bv42 contained a 0.9 kilobase (kb) DNA insert
that was excised as a single fragment with Eco RI. A double
restriction digest with Sma I and Kpn I excised a 0.96 kb
fragment that migrated just above the Eco RI fragment in
agarose gels. Sma I and Kpn I cut pBluescript SK(-) DNA
within the polylinker sequence on either side of the Eco RI
site and a non-recombinant fragment would be only 0.07 kb.
Digestion with other restriction enzymes (Hind III, Xho I,
Pvu II, Bam HI, Bgl I, and Pvu I) indicated the presence of
a 0.9 to 1.0 kb insert.
Characterization of recombinant protein frBv42) In
immunoblots using R-914 and Babb 35A4, the recombinant B.
bovis protein expressed in E. coli migrated in SDS-
polyacrylamide gels at an apparent molecular weight of 45
kDa (Lane 6, Figs. 3-2 A and B), slightly larger than the
native glycoprotein (Lane 7, Figs. 3-2 A and B). Neither
antibody reacted with proteins produced by E. coli (XLl-Blue
host strain) without the phagemid or E. coli containing an
unrelated recombinant Bluescript phagemid. In addition, the
recombinant Bv42 protein could not be detected in
immunoblots probed with normal rabbit sera or isotype
control monoclonal antibody (Figs. 3-2 A and B, Lanes 1-5).


59
The relative molecular weight of recombinant Bv42 was
confirmed by SDS-PAGE after R-914 antisera and Babb 35A4
monoclonal antibody were used to immunoprecipitate from
bacterial antigen that had been metabolically labeled in
vitro with 35S-methionine.
Antibodies against rBv42 recognize native Bv42 Following
immunization with rBv42, all four rabbits and one of two
cattle produced antibodies that immunoprecipitated a 42 kDa
molecule from native B. bovis antigen (Fig 3-3). The native
protein precipitated by antibodies raised against the
recombinant protein could be metabolically labeled in
infected erythrocyte cultures with 35S-methionine or 3H-
glucosamine and was radioiodinated on live merozoites by a
surface specific method (Fig 3-4). With each radiolabeling
method, the precipitated protein comigrated in SDS-PAGE with
a 42 kDa parasite protein immunoprecipitated using the
merozoite surface reactive monoclonal antibody Babb 35A4
(Fig. 3-4).
Antibodies from a rabbit immunized with rBv42 were used
to deplete a B. bovis antigen preparation of the native 42
kDa surface protein defined by Babb 35A4. A single
immunoprecipitation using R-S21 sera precipitated a 42 kDa


60
protein from 35S-methionine labeled parasite antigen (Fig.
3-5, Lane 2). A second immunoprecipitation from the
resulting supernatant using sera from the same rabbit showed
that none of the 42 kDa protein recognized by antibodies in
R-S21 sera remained (Fig. 3-5, Lane 3). A radiolabeled
protein was also not detected when the depleted supernatant
antigen was subsequently precipitated using Babb 35A4 (Fig.
3-5, Lane 4). In contrast, the same antigen preparation not
previously depleted with antibodies against rBv42 contained
a 42 kDa protein that could be immunoprecipitated using Babb
35A4 (Fig. 3-5, Lane 5).
Discussion
Bv42 is the immunodominant B. bovis merozoite surface
antigen, preferentially recognized by antibodies from cattle
protected against babesiosis [98]. This 42 kDa integral
membrane glycoprotein can be surface radiolabeled and was
originally defined by monoclonal antibody Babb 35A4 that
bound to an exposed epitope on live merozoites [72], In
addition, Bv42 has previously been shown to be species
specific and to contain epitopes conserved in at least four
geographic isolates [96 and unpublished data]. These
features make Bv42 a strong candidate for inclusion into a
subunit vaccine against B. bovis.


61
The marked immunodominance of Bv42 and conservation of
epitopes in multiple isolates suggests also that this
protein will be a valuable diagnostic reagent. Affinity
purified rBv42 or a synthetic peptide corresponding to the
immunodominant region of the native protein would provide a
B. bovis-specific target antigen for use in an antibody
based diagnostic test [96].
B. bovis cDNA cloned into Lambda ZAP II and
subsequently subcloned into Bluescript SK(-) phagemid
encoded a recombinant parasite protein that is a faithful
immunologic replica of the native molecule. The recombinant
protein bears the Bv42 surface epitope defined by the
monoclonal antibody Babb 35A4, and antisera generated
against the recombinant protein immunoprecipitated the 42
kDa native B. bovis protein. It was important to prove
definitively that the 42 kDa protein precipitated by
antibodies raised against the recombinant was the same 42
kDa surface protein defined by Babb 35A4. Both antigens
could be metabolically labeled in vitro with 3H-glucosamine
and surface labeled on live merozoites with 125I, techniques
by which a limited number of parasite proteins are
radiolabeled [98]. More directly, however, antibodies
against rBv42 bound to and removed the glycoprotein


62
recognized by Babb 35A4 from a preparation of native
antigen.
Based on its molecular weight, the recombinant protein
is predicted to include greater than 95% of native Bv42. In
SDS-polyacrylamide gels, recombinant Bv42 migrated just
above the native protein at a relative molecular weight of
45 kDa. Expression of a gene cloned into the unique Eco RI
site of Lambda ZAP II should produce a fusion protein
containing 3.9 kDa of vector encoded polypeptide
(Stratagene). Since Bv42 would not be glycosylated in a
prokaryotic vector, rBv42 would likely migrate at a lower
relative molecular weight than the native glycoprotein.
Therefore, the 45 kDa rBv42 described here is predicted to
include at least 41 kDa of the 42 kDa native molecule.
Restriction enzyme mapping of isolated phagemid DNA
confirmed that the cDNA insert was cloned into the unique
Eco RI site of the vector. A 0.9 kb fragment was excised
using Eco RI and restriction enzymes that cut within the
multiple cloning site on either side of the Eco RI site. An
insert of this size should encode a protein of only about 30
kDa, but many parasite antigens migrate anomalously in SDS-
PAGE at higher molecular weights than predicted due to
highly repetitive primary sequences [100]. These repetitive
regions also tend to be immunodominant and may explain the


63
marked antibody response to Bv42 in cattle infected with B.
bovis [101]. The apparent discrepancy between the size of
the insert and the relative molecular weight of the
recombinant protein in SDS-PAGE will be resolved by
sequencing this gene.
The cloning of Bv42 will now enable testing of the
hypothesis that a monospecific immune response against this
merozoite surface antigen will protect cattle against
babesiosis. The recombinant protein expressed in E. coli
contains at least one epitope exposed on live merozoites and
has already been shown to induce antibody that recognizes
native Bv42. That recombinant protein is being isolated by
immunoaffinity chromatography and will be used to immunize
cattle prior to challenge with B. bovis. In addition, the
gene for Bv42 will be subcloned into vaccinia virus for
testing as an immunogen. Expression in vaccinia virus
should result in appropriate glycosylation and folding of
rBv42, thereby preserving potentially important epitopes
contributed by carbohydrate moieties or tertiary structure
[73].


64
Babb 35A4 23.3.16
fe
23.10.36 IrG 2a
9
FIGURE 3-1 E. coli containing the recombinant phagemid
express a protein recognized by two surface reactive
monoclonal antibodies against native Bv42 and by rabbit
anti-serum (R-914) that is monospecific for Bv42. Colony
lifts from XL-1 Blue host strain bacteria (right) or XL-1
Blue containing the recombinant phagemid (left) were probed
with three monoclonal antibodies that react with the surface
of live merozoites (Babb 35A4, 23.10.36, and 23.3.16), an
IgG2a monoclonal antibody isotype control, or R-914 anti
serum. As a positive control, R-914 was also used to
detected B. bovis-infected erythrocytes that were spotted
onto nitrocellulose membranes at lOyl ul, 105/1 ul, 10A/1
ul, and 103/liil (left to right).


FIGURE 3-2 E. coli containing the recombinant Bv42
phagemid express a 45 kDa protein bearing the surface
exposed epitope defined by monoclonal antibody Babb 35A4.
Antigen was collected from the XLl-Blue E. coli host strain
(Lanes 1 and 4), XLl-Blue containing a recombinant
Bluescript SK(-) phagemid encoding an Anaplasma margnale
gene (Lanes 2 and 5), and XLl-Blue containing the rBv42-
Bluescript phagemid (Lanes 3 and 6). Bacterial antigen
(Lanes 1-6) or B. bovis iRBC antigen (Lane 7) were
transblotted and probed with normal rabbit sera (Fig. A,
Lanes 1-3), monospecific rabbit anti-Bv42 sera R-914 (Fig.
A, Lanes 4-7) an IgG2a isotype-control monoclonal antibody
control (Fig. B, Lanes 1-3), or Babb 35A4 (Fig. B, Lanes 4-
7) .


1 2 3 4 5 6 7
A.
66
-92.5
-69
-46
-30
-14.3
1 2 3 4 5 6 7 B.
-92.5
-69
-30
14.3


67
1 2
3 4 5 6 7 8 9
10 11 12 13 14
-200
FIGURE 3-3 Antibodies against the recombinant Bv42
protein immunoprecipitate a 42 kDa native protein from j5S-
methionine labeled B. bovis antigen. Metabolically labeled
protein was immunoprecipitated using pre-immunization sera
(odd numbered lanes) or post-immunization sera (even
numbered lanes) from rabbits R-S19 (Lanes 3,4), R-S20 (Lanes
5,6), R-S21 (Lanes 7,8), and R-S22 (Lanes 9,10) and cattle
B-770 (Lanes 11,12) and B-783 (Lanes 13,14). Parasite
antigen was also precipitated using sera from control
rabbits immunized with lysates of E. coli host strain XL1-
Blue (R-126, Lane 2) or XLl-Blue containing an A. margnale
recombinant phagemid (R-108, Lane 1).


A.
B.
68
FIGURE 3-4 Bovine antibodies against the recombinant
Bv42 protein immunoprecipitate a 42 kDa native protein that
is metabolically labeled with 3H-glucosamine, surface
labeled on B. bovis merozoites, and co-migrates with the
surface protein defined by Babb 35A4. A. Parasite antigen
was labeled in vitro with 3H-glucosamine and
immunoprecipitated using pre-immunization sera from steer B-
783 (Lane 1), post-immunization sera from B-783 (Lane 2),
Babb 35A4 (Lane 3), or an IgG2a control monoclonal antibody
(Lane 4). B. Radioiodinated merozoite surface proteins
were immunoprecipitated using pre-immunization sera from B-
783 (Lane 1), post-immunization sera from B-783 (Lane 2), an
IgG2a control monoclonal antibody (Lane 3), and Babb 35A4
(Lane 4). Arrow indicates the ^5I-labeled 42 kDa native B.
bovis protein. With both antigen preparations, identical
results were obtained using rabbit anti-rBv42 sera (data not
shown). Immunoprecipitations using sera from control
rabbits immunized with E. coli (R-126) or E. coli containing
a control phagemid (R-108) revealed no specific bands on
SDS-PAGE.


200
-92.5
-69
-46
-30
14.3
FIGURE 3-5 Antibodies against the recombinant Bv42
protein bind to and remove the glycoprotein recognized by
Babb 35A4 from a preparation of native antigen. 35S-
methionine labeled antigens of B. bovis contain a 42 kDa
protein that is immunoprecipitated using R-S21 rabbit anti-
rBv42 antibodies (Lane 2) or Babb 35A4 (Lane 5), but not
using R-S21 pre-immunization sera (Lane 1) or an IgG2a
control monoclonal antibody (Lane 6). One
immunoprecipitation using R-S21 antibodies completely
removes the 42 kDa protein from an antigen preparation so
that it can no longer be immunoprecipitated using additional
R-S21 antibodies (Lane 3) or Babb 35A4 monoclonal antibody
(Lane 4).


CHAPTER 4
SUMMARY \ CONCLUSIONS
Merozoite surface antigens are potential targets of the
protective immune response to Babesia bovis and, therefore,
candidates for an improved vaccine. Antibody directed
against surface exposed regions of merozoite antigens is
likely to inhibit recognition and invasion of erythrocytes.
Alternatively, antibody against the merozoite surface could
opsonize the parasite for phagocytosis or, in conjunction
with complement, mediate merozoite lysis.
Using antibodies from cattle which are protected
against babesiosis, I have identified eight surface-
radiolabeled merozoite proteins from a cloned, pathogenic B.
bovis isolate. Among these eight surface proteins, the 55
and 42 kilodalton molecules were glycoproteins that could be
biosynthetically labeled with 3H-glucosamine. The 42 kDa
glycoprotein, designated Bv42, could also be labeled with
3H-myristic acid and partitioned into the detergent phase in
Triton X-114 extracts, indicating that it was an integral
membrane protein and suggesting that it was anchored by a
glycosylphosphatidylinositol moiety. This information may
70


71
be important for the presentation of Bv42 as an immunogen.
Native or recombinant protein may need to be presented in
the context of a membrane to generate antibody that reacts
with the antigen as it is expressed on the surface of the
merozoite.
Antibody mediated protection against B. bovis
merozoites most likely requires a high level of circulating
antibody to ensure antibody-merozoite binding during the
parasite's transient extracellular phase. Antibodies in
diluted sera from protected cattle selectively recognize the
120, 85, 55, and 42 kDa surface proteins. Only the 42 kDa
integral membrane glycoprotein Bv42 is reactive with serum
antibodies diluted > 1:16,000. I hypothesized that the
immunodominant proteins, especially Bv42, were important to
the induction of the protective immune response and set out
to test that hypothesis by first cloning the genes encoding
one or more of these candidate immunogens. Cloning and
expression will provide parasite antigen in sufficient
quantity and purity to test in immunization trials.
A 0.9 kb cDNA fragment encoding the 42 kDa
immunodominant merozoite surface antigen Bv42 was cloned
into the unique Eco RI site of the bacteriophage vector
Lambda ZAP II. Expression of recombinant DNA in the
multiple cloning site of the lac Z gene of Lambda ZAP II


72
should produce a fusion protein containing only 3.9 kDa of
vector encoded polypeptide. The 45 kDa recombinant B. bovis
protein expressed in Escherichia coli accounted for greater
than 95% of the molecular weight of the native 42 kDa
glycoprotein and, importantly, included the epitope(s)
defined by monoclonal antibodies that react with the surface
of live merozoites.
Antibodies were prepared against the recombinant
Babesia protein to prove definitively that the cloned gene
encoded Bv42 and to show that antibodies directed against
the recombinant protein recognize the native parasite
protein. This second point, that the recombinant protein
functions as an immunologic replica of the native protein,
is critical if recombinant Bv42 (rBv42) is to be tested as
an immunogen for babesiosis. Antibodies from animals
immunized with rBv42 immunoprecipitated the 42 kDa
glycoprotein from surface labeled and metabolically labeled
B. bovis. Further, antibodies against the recombinant
protein bound to and removed the 42 kDa protein recognized
by surface reactive monoclonal antibody Babb 35A4 from a
preparation of native, methionine labeled parasite antigen.
A successful immunization trial using an isolated B.
bovis antigen would focus the efforts to develope an
improved vaccine. In addition, however, the work to isolate


73
the genes for B. bovis antigens and develop monospecific
reagents will help answer important questions about the
basic biology of Babesia spp. Those questions will include
the biologic function of merozoite surface antigens, the
extent of antigenic diversity among different geographic
isolates, the role, if any, of antigenic variation within a
single clone of parasites, and the genetic mechanisms
involved in the expression and modification of these
important parasite molecules.


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BIOGRAPHICAL SKETCH
Stephen Alan Hines was born to Della Maxene Hanna Hines
and Earl Thomas Hines on March 5, 1955, in Bowling Green,
Kentucky. He received a Bachelor of Arts in May, 1977, from
Miami University in Oxford, Ohio. On June 12, 1981, Steve
graduated as a Doctor of Veterinary Medicine from Ohio State
University, College of Veterinary Medicine. The following
day he married Dr. Melissa Gail Trogdon, also a
veterinarian. At that time Steve inherited Riley, a once-
in-a-lifetime dog.
After a year of dairy and small animal practice, Steve
moved with Melissa to Gainesville, Florida, to start an
anatomic pathology residency at the University of Florida's
Veterinary Medical Teaching Hospital. With his residency
completed in July, 1984, Steve entered graduate school in
the College of Medicine, University of Florida. In
September, 1985, Steve passed his veterinary pathology
specialty boards. Six months later, he changed major
advisors to work on the immunology and molecular biology of
veterinary hemoparasites, in particular Babesia bovis. with
Guy Palmer. When Guy left Florida in May, 1988, to be
85


86
closer to mountains, Steve moved to Terry McElwain's
laboratory to continue his dissertation work on Babesia. In
June, 1989, Terry also left Florida to return to the same
place Guy went. Soon Steve and Melissa will be following
Terry and Guy to the mountains to faculty positions at
Washington State University, that is. It's been great being
a gator, now let's try being a cougar!
After we're gone, try to preserve the natural beauty of
Florida. It's worth fighting for.


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for
the deqree of Doctor of Philosophy.
F. McElwain, Chair
L
Terry
Assistant Professor of Veterinary
Medicine
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for
the degree of Doctor of Philosophy.
Lindsey/6utt-Fletcher,Cochair
Associate Professor of Pathology
and Laboratory Medicine
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for
the degree of Doctor of Philosophy/, /-n .
Guy H. Palmer
Assistant Professor of Veterinary
Medicine
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for
the degree of Doctor of Philosophy.
Anthony/F. Barbet
Associate Professor of Veterinary
Medicine


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for
the degree of Doctor of Philosophy.
-JO
Edward Wakeland
Associate Professor of Pathology
and Laboratory Medicine
This dissertation was submitted to the Graduate Faculty of
the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
December, 1989
CHb:
Dean, Colleg
e of Medicine
Dean, Graduate School


Full Text
UNIVERSITY OF FLORIDA



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