Title of Invention

" A COMPOSITION COMPRISING ISOLATED, METABOLICALLY ACTIVE, ATENTUATED PLASMODIUM SPOROZOITES SUITABLE FOR PARENTERAL NON INTRAVENOUS ADMINISTRATION OF AT LEAST ONE VACCINE DOSE TO A MAMMALIAN OR HUMAN HOST"

Abstract The invention comprises a novel method for protecting subjects against malaria. The method of the invention invoices inoculation with attenuated sporozoites. and in particular, but not limited 10 subcutaneous, intramuscular, intradermal, mucosal, submucosal, and cutaneous administration.
Full Text
METHOD FOR THE PREVENTION OF MALARIA
CROSS REFERENCE TO RELATED APPLICATIONS
[001] This application is based on and claims the benefit of U.S. Provisional Application
S.N. 60/427,91 l, filed November 20, 2002. The entire disclosure of this provisional application
is relied upon and incorporated by reference herein.
FIELD OF THE INVENTION
[002] This application relates to preventing malaria by administering a vaccine. More
particularly, this invention relates to a vaccine against malaria infection comprising the
administration of attenuated sporozoites to a human or animal.
INTRODUCTION AND DESCRIPTION OF THE PRIOR ART
[003] Malaria is a disease that affects 300-500 million people, kills one to three million
individuals annually, and has an enormous economic impact on people in the developing world,
especially those in sub Saharan Africa [1, 2]. Plasmodium falciparum accounts for the majority
of deaths from malaria in the world. The World Tourist Organization reported that of the nearly
700 million international tourist arrivals recorded worldwide in 2000, approximately 9 million
were to West, Central or East Africa, 37 million were to South-East Asia, 6 million to South Asia
and 10 million to Oceania [3]. It is estimated that more than 10,000 travelers from North
America, Europe, and Japan contract malaria per year. For more than 100 years during every
military campaign conducted where malaria was transmitted, the U.S. forces have had more
casualties from malaria than from hostile fire. An estimated 12,000,000 person days were lost
during World War II and 1.2 million during the Vietnam conflict due to malaria [4].
[004] Transmission of the parasite Plasmodium (the protozoan parasite causing malaria)
occurs via the bite of infected female Anopheles mosquitoes, which are active from dusk to
dawn. Sporozoites migrate from the bite site to the liver via the blood stream, where they
multiply within hepatocytes, producing, in the case of P. falciparum, 10,000-40,000 progeny per
infected cell. These liver stage parasites express a set of antigens which are not expressed in
sporozoites. This new generation of parasites re-enters the blood stream as merozoites,
expressing a set of antigens which are different from those expressed during the sporozoite and
early hepatic stages, and invade erythrocytes, where additional multiplication increases parasite
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numbers by approximately 10 to 20 fold every 48 hours. Unlike the five to ten day development
in the liver, which does not induce any symptoms or signs of illness, untreated blood stage
infection causes hemolysis, shaking chills, high fevers, and prostration. In the case of P.
falciparum, the most dangerous of the four species of Plasmodium that infect humans, the
disease is complicated by disruption of microcirculatory blood flow and metabolic changes in
vital organs such as the brain, kidney and lung, frequently leading to death if not urgently
treated.
[005] An effective vaccine against P. falciparum malaria remains one of the great
challenges of medicine. Despite over one hundred years of effort, hundreds of millions of
dollars in research, lifelong sacrifice from dedicated physicians and scientist and many
promising experimental vaccines, there is no marketed vaccine to alleviate one of the great
infectious scourges of humanity. A generation ago, public health initiatives employing
chloroquine, DDT and vector control programs seemed poised to consign falciparum malaria to
insignificance as a worldwide menace. The lack of an effective vaccine complicated these
efforts, but sustainable control seemed imminent.
[006] The promise of impending success was short lived and the reasons for failure were
multi-factorial. The parasites grew increasingly resistant to highly effective and affordable anti-
malarial medications, vector control measures lapsed, and trans-migration, war and economic
disruption became increasingly more common in endemic areas of the developing world. As a
result, falciparum malaria has resurged, annually placing 2.5 billion humans at risk, causing 300-
900 million infections, and killing 1-3 million people. Of the many social, economic,
environmental and political problems that afflict the developing world, P. falciparum malaria is
increasingly seen as both a root cause and cruel result of these inequities, and is a singular
impediment to solving these complex problems. Controlling falciparum malaria in the
developing world may be possible without an effective vaccine. In practice, given social,
political and economic realities, the inventors believe that a vaccine may be an essential
component of a sustainable control program, and will be required for a global eradication
campaign.
[007] It is in this context that the modern period of malaria vaccine development has been
particularly frustrating. Since the early 1980's, breathtaking technological advances in
molecular biology and medical science have occurred. These advances accelerated the
identification of stage-specific P. falciparum proteins and epitopes, and host immune
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mechanisms and responses. This knowledge was translated into a range of novel vaccine
candidates [5,6]. In one sense, this modem period has been the golden age of malaria vaccine
research and human testing. However, in spite of the Herculean efforts of malaria researchers,
the majority of these vaccines have failed to provide any protective immunity in humans — with
only one demonstrating reproducible short term protection against infection in 40%-70% of
recipients [7-9].
[008] Given enough time and resources, these vaccine strategies, or others yet to be
developed, may ultimately lead to a robust vaccine. However, at a recent Keystone meeting,
"Malaria's Challenge: From Infants to Genomics to Vaccines" [6], the attendees were polled as
to when they thought a malaria vaccine might be "launched" as a commercial product. Many in
the room indicated that they thought the first vaccine would not be launched until 2016-2025.
The leader of Glaxo Smith Kline's (GSK) efforts to develop a recombinant P. falciparum
circumsporozoite protein (PfCSP) vaccine voiced the most optimism. It was indicated that if all
went well, this single protein vaccine could be "launched" in 7-8 years (2009-2010). Given that
GSK and the U.S. Army have been working on a recombinant protein PfCSP vaccine since the
1984 cloning of the PfCSP [10], and that many malariologists express concern as to whether a
single protein vaccine will be adequate to sustainably control malaria, this time line of more than
25 years for development of a single protein vaccine places a chillingly realistic perspective on
the possibilities for developing vaccines that will truly reduce the burden of this disease.
[009] Protective Immunity After Immunization with Radiation Attenuated
Sporozoites: In, 1967 Nussenzweig repotted that intravenous administration of radiation,
attenuated P. berghei sporozoites to A/J mice protected the mice against challenge with
infectious P. berghei sporozoites [11]. These rodent studies provided the impetus for human
studies, and by the early 1970s, two groups established that immunizing human volunteers with
the bites of irradiated mosquitoes carrying P. falciparum sporozoites in their salivary glands
could protect volunteers against challenge with fully infectious P. falciparum sporozoites [12-
19]. These studies demonstrated that a malaria vaccine offering sterile protective immunity was
possible. However, the only way to produce sporozoites at that time was to infect a volunteer
with P. falciparum, treat the volunteer with doses of chloroquine to suppress but not eliminate
the parasite, allow gametocytes to develop, and then feed mosquitoes on these volunteers. Even
if one could produce sporozoites in adequate numbers by this method, it was considered
clinically, technically and logistically impractical to immunize humans with an irradiated
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sporozoite vaccine. In large part this was because the sporozoites had to be delivered alive,
either by the bite of infected mosquitoes, or by intravenous injection as was done with mice. The
scientists active in the field concluded that other routes of immunization would not provide
adequate or comparable protection as compared to immunization by intravenous injection or by
the bite of infected mosquitoes; in essence ruling out the use of attenuated sporozoites as a
vaccine from their perspective. The published views of several such scientists are quoted below.
[010] "This observation corroborates previous reports (Nussenzweig, Vanderberg and
Most, 1967 and 1969) and extends their findings. Groups of mice immunized by other parenteral
routes (i.m., i.p., and i.e) exhibited an overall level of protection much lower than the i.v.
immunized mice." [20]
[011] "These studies have confirmed a previous report which demonstrated that
intramuscularly injected irradiated sporozoites of P. berghei are far less effective than these
injected intravenously in protectively immunizing mice against sporozoite-induced malaria...The
chief limitation preventing an extension to human trials was the requirement for intravenous
immunization a procedure posing unacceptable medical risks," (In the study referred to in this
quotation, protection by the intramuscular route ranged between. 11% and 42% and protection
by the subcutaneous route was 0%) [21].
[012] "It was further shown that of the various routes of immunization used in vaccination
attempts in rodents (i.m., i.v., subcutaneous, per os, etc.) the intravenous route gave the highest
degree of protection and most reproducible results. The only other very effective route of
immunization is by the bite of infected, irradiated mosquitoes." [22]. In this 1980 review, "Use
of Radiation-attenuated sporozoites in the Immunoprophylaxis of Malaria," Dr- Nussenzweig
goes on to discuss the potential for developing a sporozoite malaria vaccine, and concludes, "In
conclusion, recent findings appear to indicate that we now have the necessary powerful tools
which should provide the means to clarify the mechanism of sporozoite-induced immunity and to
isolate the protective antigens. Under these conditions, the various obstacles to the development
of a sporozoite vaccine for malaria appear to be surmountable, hopefully in the not too remote
future." Dr. Nussenzweig does not discuss the idea of utilizing a whole attenuated sporozoite
vaccine as a reasonable alternative, only the use of sporozoites to provide the components of a
vaccine that induces immunity against the sporozoite stage.
[013] In 1980 after nearly 15 years of work on the irradiated sporozoite vaccine model, it
was concluded by the unquestioned leader in the field, Dr. Nussenzweig, that the route to a
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vaccine lay through modem science; understanding immunologic mechanisms of protection and
the antigenic targets of those protective immune responses, and constructing a "subunit"
sporozoite vaccine. From then onwards there was essentially no mention or discussion in the
literature of trying to develop an attenuated whole parasite sporozoite vaccine as a practical
vaccine for humans for many reasons, not the least of which was that despite these 15 years of
research, no scientists had discovered a reasonable approach to administering sporozoites other
than by intravenous administration or by the bite of infected mosquitoes.
[014] There was also no further work to develop an attenuated sporozoite vaccine,
because the sporozoites would have to be raised in aseptic mosquitoes, aseptically purified, and
suitably preserved and reconstituted prior to administration, and after such treatment would still
have to be able to elicit protective immune responses when administered.
[015 ] Potential solutions to parts of the problems of production, though not recognized at
the time as being related to developing an attenuated sporozoite vaccine, were being reported. In
1975, a method for culturing P.falciparum in vitro was reported [23, 24], followed in 1982 by a
method for producing gametocytes from these cultures [24]. In 1986, it was reported that humans
could be infected by the sporozoites produced in mosquitoes that had fed on these in vitro
cultures [26]. There was therefore a way to produce sporozoites without the difficulties of in vivo
production of gametocytes in humans. These developments on their own were not adequate to
overcome all of the obstacles to development of attenuated sporozoite vaccine. There was not a
way to produce enough of the sporozoites or produce and process the sporozoites under
conditions that met regulatory standards. Furthermore, there were no data indicating that
properly produced and processed sporozoites could be administered successfully in a clinically
acceptable and practical manner.
[016] Thus, following the failure of the malaria scientific community to discover a
method to deliver attenuated sporozoites in a clinically acceptable and practical manner
sufficient to achieve high level protection, the attenuated sporozoite vaccine was dropped from
clinical consideration and the community as presaged by Dr. Nussenzweig'(paragraph [012]
above) embraced modem molecular science in the hope of developing a vaccine. Several
promising developments launched the modern era of malaria sub-unit vaccine development. A
monoclonal antibody against the major surface protein of sporozoites, the circumsporozoite
protein (CSP), had been produced and shown to protect mice in passive transfer experiments
[27]. Additionally, the gene encoding the PfCSP protein had been cloned and sequenced [10].
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Coincidentally, the first purified recombinant protein vaccine, the hepatitis B surface antigen
vaccine, was developed and marketed [28]. The weight of evidence and trends in vaccine
science seemed to offer malaria researchers a roadmap to quickly develop a human malaria
vaccine. Since it was considered impractical to produce and administer the sporozoite vaccine,
returning to an attenuated whole parasite vaccine seemed unnecessary and dated, and all
subsequent efforts focused on the promise of sub-unit vaccines.
[017] In 1987 when the first recombinant protein [29] and synthetic peptide [30] vaccines
did not prove to be as protective as expected, instead of considering the development of an
attenuated sporozoite vaccine which was considered impossible to produce and administer,
scientists focused on understanding the immune mechanisms responsible for protective
immunity, and the antigenic targets of these protective immune responses, and developing
subunit vaccines and vaccine delivery systems that induced such protection. Much of this basic
work was carried out in the P. berghei and P. yoetii rodent model systems. This rodent malaria
work provided important insights into immunologic mechanisms and antigenic targets of
irradiated sporozoite vaccine-induced protection and led to the development of a number of
candidate vaccines [31-33]. None of these studies which were conducted after the cloning of the
gene encoding the P. falciparum circumsporzoite protein (PfCSP) in 1984 through the end of the
millenium suggested the possibility of developing a human irradiated whole sporozoite vaccine,
because none of the investigators thought it was possible to produce or administer such a vaccine
in a practical manner. Interestingly, sub-unit (recombinant protein, synthetic peptide,
recombinant virus. DNA plasmid) vaccine formulations have been shown to produce excellent
protection in mice, but nothing comparable in humans. In contrast the protection in mice by
intravenous administration of irradiated sporozoites [11] led to human studies, that demonstrated
that exposure to the bites of irradiated mosquitoes with P. falciparum sporozoites in their
salivary glands induced protection [34].
[018] In 1989, after a number of disappointing clinical trials of sub-unit PfCSP vaccines,
immunization of volunteers by the bites of mosquitoes carrying P. falciparum sporozoites in
their salivary glands and then attenuated by exposure in vivo to gamma radiation was begun at
the Naval Medical Research Institute later Naval Medical Research Center (NMRI later NMRC)
and Walter Reed Army institute of Research (WRAIR). The goal of this research was to better
delineate the clinical characteristics and requirements that led to protecting humans with the,
irradiated sporozoite vaccine, assess the protective immune responses elicited in humans, and
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identify the antigens and epitopes on those proteins that elicited immune responses in humans. It
was never a consideration to develop irradiated sporozoites as a human vaccine, as it was
considered completely impractical and technically unfeasible to produce such, a vaccine as well
as to administer such, a vaccine. Preliminary clinical results and extensive immunological assay
results from these studies were published [35-41]. These immunological studies combined with
those of others on this subject [42-48] increased our understanding of the immunological
responses in humans immunized with radiation attenuated P. falciparum sporozoites. However,
there waS no consideration or mention of trying to develop an attenuated sporozoite vaccine.
[019] The results of the first 10 years' clinical experience with these immunizations and
challenges were recently reported, and combined with all the published clinical reports of
immunizing humans with irradiated Plasmodium sporozoites [34] from the University of
Maryland (1970's, late 1980's and early 1990's), and the Rush - Presbyterian - StLuke's Medical
Centre in Chicago and the Naval Medical Research Institute in the 1970's [12-19, 34]. A number
of observations arose from the analysis that was conducted.
[020] A). There was a dose response in regard to protective immunity among volunteers
challenged by the bite of 5-14 infected mosquitoes. Thirteen of 14 volunteers (93%) immunized
by the bites of greater than 1000 infected, irradiated mosquitoes were protected against
developing blood stage P. falciparum infection when challenged within 10 weeks of their last
primary immunization. There were 35 challenges of these volunteers and there was complete
protection against development of blood stage infection in 33 of the 35 challenges (94%). Four
of 10 volunteers (40%) immunized by the bite of greater than 200 and less than 1000 infected,
irradiated mosquitoes were protected against developing blood stage P. falciparum infection
when challenged within 10 weeks of their last primary immunization, a significantly lower level
of protective immunity than among volunteers immunized with> 1000 infective bites (p=
0.0088, Fisher's exact test, 2-tailed). There were 15 challenges of the volunteers immunized
with less than 1000 infective bites, and there was complete protection against development of
blood stage infection in 5 of the 15 challenges (33%), a significantly lower level of protective
immunity than among volunteers immunized with > 1000 infective bites (p= 0.000015, Fisher's
exact test, 2-tailed).
[021] B). Protective immunity lasted for at least 42 weeks (10.5 months). Five of 6 of
the above 14 volunteers when challenged from 23 to 42 weeks (23, 36, 39, 41, and 42 weeks)
after their last primary or secondary immunization were protected against experimental
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challenge. Except for a single challenge of one volunteer five years after last immunization (not
protected), there were no other challenges assessing longevity of protective immunity.
[022] C). Protection was not strain specific. Four volunteers were challenged with
isolates of P. falciporum different than, the isolates with which they were immunized. The four
volunteers were completely protected in seven of seven such challenges with different isolates of
P. falciparum.
[023] D). Immunologic memory lasts for at least 5 years. A volunteer who had been
exposed to the bite of 1601 irradiated infected mosquitoes, and protected when challenged 9 and
42 weeks after last exposure, was not protected when re-challenged 5 years after last exposure to
to irradiated, infected mosquitoes. He was treated for his malaria, boosted by exposure to 147
irradiated, infected mosquitoes, and re-challenged by exposure to the bite of 5 non-irradiated
mosquitoes infected with P. falciparum sporozoites. This volunteer was protected against that
infectious challenge [34], demonstrating that the protective immunity was boostable with a
single exposure to irradiated sporozoites.
[024] Thus, protection was achieved in greater than 90% of challenge experiments after
greater than 1000 mosquito bites, lasted for at least 10.5 months, and was not P.falciparum
isolate (strain) specific.
A "sub-unit" vaccine demonstrating this level of protective efficacy in human subjects
would be recognized as a major breakthrough. Though it was routinely observed that protection
resulted from this experimental irradiated sporozoite vaccine, the sheer power of attenuated
sporozoites remained unrecognized until after completion of the careful analysis necessary to
publish this report. Interestingly, when these results were presented by one of us (SLH) at the
Keystone meeting in March 2002, "Malaria's Challenge: From Infants to Genomics to
Vaccines," they were considered interesting, but no one in the audience even raised the idea that
this approach should be pursued as viable malaria vaccine, because all thought the vaccine to be
impractical to produce and impossible to administer. This view is still widely held in the
scientific community. In a recent publication in Nature magazine (October 2, 2003) [49], the
director of clinical trials at the Naval Medical Research Center Malaria Program stated, "The
barriers have seemed sufficiently daunting that no one has been willing to give it a try," and a
malaria vaccine expert from the University of Oxford in the United Kingdom, stated, "It's a long
shot ....It's worth a try, although the odds are heavily stacked against him." In contrast, the
inventors believed that it was possible to make such a vaccine, but there were several critical
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questions that had to be answered before moving into cGMP manufacturing and clinical trials.
These are outlined in a recent publication [50] One of the most critical questions was whether
one can administer attenuated sporozoites by a route that is practical for a human vaccine?
SUMMARY OF THE INVENTION
[025] Heretofore, it had been considered impractical to immunize humans with attenuated
Plasmodium species sporozoites, because the sporozoites had to be delivered by the bite of
infected irradiated mosquitoes for immunization, or by intravenous injection, as this was what
had been done previously with humans and mice respectively, and was accepted by the scientific
community as the only way to achieve high level protective immunity.
[026] It has been theorized that when properly irradiated sporozoites are delivered by
mosquito bite or intravenous injection, they pass through the bloodstream to the liver, invade
hepatocytes, partially develop, and then arrest development, never developing to the mature liver
sehizont, which ruptures, and releases merozoites which cause infection of erythrocytes, and the
disease known as malaria. Thus, they are attenuated. Data indicate that in order to elicit adequate
protective immune responses, the parasites must invade hepatocytes, partially develop, and
express new proteins that are the targets of protective immune responses, particularly CD8 T
cells.
[027] The inventors theorized that there is a direct correlation/assocation between the
infectivity of a preparation of unirradiated sporozoites and their capacity to elicit protective
immunity when they are attenuated. Furthermore, we theorized that there is a direct
correlation/assocation between the infectivity of unirradiated sporozoites when administered by a
particular method, and the capacity of those sporozoites when irradiated and delivered by that
method to elicit protective immunity.
[028] The present invention described herein was discovered in response to asking the
question, can one administer the attenuated sporozoites by a route that is practical for a human
vaccine?
[029] This question was addressed using the P. yoelii rodent malaria parasite, not the P.
berghei rodent malaria parasite, which had been studied previously in all reports cited above (11,
20-22). The P. berghei model system was used to establish that irradiated sporozoites protect A/J
mice, and this led to the human studies demonstrating that exposure to irradiated P.falcipatrum
infected mosquitoes protects humans. The P. berghei system was also used to prove to the
scientific community that intramuscular, subcutaneous and other non-intravenous routes of
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administration of irradiated sporozoites are not adequately protective in mice (20-22). In fact
after subcutaneous administration of radiation attenuated sporozoites protection was 0% [21].
These studies which were primarily done in A/J mice led to the conclusion that it was not
possible to develop irradiated sporozoites as a practical, clinically relevant malaria vaccine for
humans. In the early to mid 1980s the Naval Medical Research Institute laboratory switched
from working with P. berghei in A/J mice to working with P. yoelii in BALB/c mice. This was
because the scientists at the Naval Medical Research Institute believed that intravenously
administered P. yoelii in BALB/c mice was more predictive of P. falciparum infection in humans
than was intravenously administered P. berghei. This was in large part because intravenously
administered P. yoelii sporozoites are so much more infectious to mice than are intravenously
administered P. berghei sporozoites. The 50% infectious dose to mice of intravenously
administered P. yoelii in BALB/c mice is approximately 100-1000 times lower than the 50%
infectious dose of P. berghei in BALB/c mice and almost certainly more comparable to the 50%
infectious dose of Plasmodium sp. parasites in primates, such as P. knowlesi in monkeys and P.
falciparum in humans than is P. berghei. In the early 1990s, approximately 10 years after the
Navy group began working with P. yoelii instead of P. berghei, after reading papers and hearing
presentations from scientists from the Navy group, Dr. Nussenzweig requested the P. yoelii
parasites used by the Navy laboratory from one of the inventors (SLH), and essentially switched
the work in her group at New York University on rodent malaria to the P. yoelii model system,
primarily working with BALB/c mice.
[030] It is important to note that all work with P. yoelii has focused on administration by
intravenous injection or mosquito bite, almost certainly because of the previous work in the P.
berghei model system described above [11, 20-22]. Furthermore, because of that work in the P.
berghei model system no one has experimented in the P. yoelii system to try to use it as a model
to develop an attenuated whole sporozoite vaccine. Immunization with irradiated sporozoites in
the P. yoelii rodent malaria system has been used by scientists for the same scientific objectives
described in 1980 by Nussenzweig [22]; to identify the immune mechanisms of protective
immunity and the antigenic targets on the parasite of these protective immune responses. For this
reason, since it has been "known" for more than 25 years that only intravenous or mosquito bite
administration of sporozoites provides the 100% protective immunity that makes the irradiated
sporozoite model so effective, these have been the routes of administration used by scientists
working in this system. The other routes (e.g. subcutaneous, intramuscular, intradermal and
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others) that would be required to make the irradiated sporozoite clinically practical and
acceptable have not been used.
[031] The inventors have discovered a method for immunizing subjects against malaria
which allows for the vaccination of large numbers of subjects with attenuated sporozoites in a
relatively short time, avoids the impracticality and potential danger of the previous methods of
bite by infected mosquitoes, or in the case of mice by intravenous injection, and which provides
protection comparable to that achieved by these prior methods.
[032] More particularly, we have discovered that effective protection against malaria can
be obtained by parentally administering a dosage of attenuated sporazoites to a subject by a route
other than intravenous injection, including, but not limited to the subcutaneous, intramuscular,
inteadermal, mucosal, submucosal, epidermal, and cutaneous routes.
DETAILED DESCRIPTION OF THE INVENTION
[033] The instant invention provides a new clinically relevant and acceptable method of
administering attenuated Plasmodium species sporozoites that makes it practical for attenuated
sporozoites to be used as a vaccine to prevent malaria in humans, mammals, avians, and other
relevant species.
[034] The invention's significant improvement over previously standard methods of
administration, administration by intravenous injection or by the bite of infected mosquitoes, of
attenuated sporozoites is that it allows for a clinically practical and safe method of administering
the vaccine that provides protection comparable to the previous standard methods.
Administration by the bite of infected mosquitoes can never be used as a vaccine for obvious
reasons, and administration by intravenous injection is a, method that is not in general use for any
vaccine, because it is a technically difficult method of administration, especially in young
children, and it is potentially dangerous because of direct injection into the bloodstream.
[035] With the present invention, the parenteral administration may be administered in the
skin (transcutaneous. epidermally, intradermally), subcutaneous tissue (subcutaneously), muscle
(intramuscularly), through the mucous membranes, or in the submucosal tissue-Preferably, the
administration is subcutaneously, intrdermally or intramuscularly.
[036] The goal of attenuation is to weaken the parasites, so that they are viable enough to
invade host cells and produce new proteins, but unable to produce a replicating asexual blood
stage infection that causes disease. Attenuation can occur in multiple ways. For example this
can occur by attenuating the parasites so that inoculated sporozoites can invade host cells,

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partially develop in these cells, and arrest development before reaching the stage compatible to a
mature hepatic stage parasite that can rupture releasing merozoites that invade erythrocytes and
cause disease. This type of attenuated parasite can be termed a metabolically active, non-
replicating parasite. Attenuation could also occur by producing parasites that can invade and
normally develop in host cells to the stage comparable to a mature hepatic stage parasite, rupture
from the host cells, but be unable to develop in erythrocytes to the point required for them to
cause disease. This could also occur by attenuating the parasites so that they can invade and
normally develop in host cells to the stage comparable to a mature hepatic stage parasite, rupture
from the host cells, but be unable to develop in erythrocytes to the point required for them to
cause significant disease. This could also occur by attenuating the parasites so that sporozoites
partially develop and produce new proteins, but arrest development before reaching the stage
comparable to a mature hepatic stage parasite that can rupture releasing merozoites that invade
erythrocytes and cause disease.
[037] While numerous methods of attenuation may be used, we have found that
attenuation by irradiation is currently preferred for producing a metabolically active, non-
replicating parasite. Attenuation of the sporozoites can be accomplished in multiple ways with
multiple dosage regimens. The attenuation can be accomplished while the sporozoites are still in
the mosquito, after they have been isolated from the mosquitoes and before interventions such as
cryopreservation, or after they have been isolated from the mosquitoes and after interventions
such as cryopreservation. The current dose of irradiation based on previous experience is
generally greater than 12,000 Rads (cGy) and less than 23,000 Rads (cGy) for Plasmodium
falciparum sporozoites with 15,000 Rads (cGy) being most commonly used [34]. One skilled in
the art will recognize that this dosage may vary from species to species or strain to strain or with
the apparatus and techniques used to irradiate the sporozoites. One skilled in the art will
recognize that the irradiation can be accomplished using numerous methods, including, but not
limited to gamma rays, x-rays, ultraviolet rays, or other subatomic particles such as electrons,
protons, or combinations of these methods.
[038] In the future, attenuation as described in paragraph [39] above may be achieved by
genetic manipulation of the parasites prior to their being introduced into the vaccine recipient.
[039] Attenuation may also be achieved by treating individuals before or after exposure to
sporozoites with drugs which prevent development of the parasites so that they can replicate in
hepatoctyes.
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[040] Attenuation may also be achieved by treating individuals before or after exposure to
sporozoites with drugs which prevent development of the parasites so that they can replicate in
erythrocytes.
[041] Attenuation may also be achieved by treating the sporozoites with chemicals which
attenuate the parasites.
[042] The means of administration may be any methods for inoculation other than by
mosquito bite or intravemous administration, such as, but not limited to injection with a single
needle and syringe, multiple needles and syringe arrays, micro-needles with one to hundreds to
thousands of pores, needleless injection by ballistic techniques, and the like. The attenuated
sporozoites may also be delivered by a transcutaneous patch, or on a particulate material, for
example, gold beads. While it is possible to achieve a level of protection with a single
inoculation, it is preferred that a series of two or more inoculations or exposures be effected.
[043] The preferred inoculant is a malaria mimunization effective amount of attenuated P.
falciparum or other Plasmodium species sporozoites. The dosage in humans per inoculation may
range from about 1,000 to 10, 000,000, although this may be varied depending on evaluation by
the practitioner or the immunogenicity /potency of the attenuated sporozoite preparations.
[044] Any Plasmodium species parasite, even if altered genetically, may be used in the
method of the invention. In one embodiment, the parasite is P. falciparum. In other
embodiments, for example, the parasite may be P vivax, P. ovale, or P. malariae. In other
embodiments it could a mixture of these parasites. In other embodiments it could be
Plasmodiium knowlesi, P. yoelii, or other Plasmodium species parasites.
[045] In one embodiment the invention provides a pharmaceutical kit comprising the
attenuated sporozoites in the delivery instrument such as a syringe.
[046] In other embodiments the invention provides a kit which includes a container such
as a vial, but not limited to a vial containing the frozen attenuated sporozoites, a container such
as a vial containing fluid to dilute the attenuated sporozoites, and the actual delivery devices,
such as a syringe and needle.
[047] In other embodiments the invention provides a kit which includes a container such
as a vial, but not limited to a vial containing the freeze-dried (lyophilizied) attenuated
sporozoites, a container such as a vial containing fluid to dilute the attenuated sporozoites, and
the actual delivery devices, such as a syringe and needle.
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[048] In other embodiments the invention provides a kit which includes a container such
as a vial, but not limited to a vial containing preserved attenuated sporozoites, a container such as
a vial containing fluid to dilute the attenuated sporozoites, and the actual delivery devices, such
as a syringe and needle.
[049] The invention further provides the use of parenteral administration of attenuated
Plasmodium species sporozoites as described herein, in the administration of a vaccine for
prevention or reduction of severity of malaria.
[050] The invention provides partial, enhanced, or full protection of a human who has not
previously been exposed to a malaria-causing pathogen, or has been exposed, but is not fully
protected. The invention may also be used to reduce the chance of developing a malaria
infection, reduce the chance of becoming ill when one is infected, reduce the severity of the
illness, such as fever, when one becomes infected, reduce the concentration of parasites in the
infected person, or to reduce mortality from malaria when one is exposed to malaria parasites. In
many cases even partial protection is beneficial. For example, a vaccine treatment strategy that
results in any of these benefits of about 30% of a population may have a significant impact on a
the health of a community and of the individuals residing in the community.
[051] A "vaccine" is a composition of matter comprising a preparation that contains an
infectious agent or its components which is administered to stimulate an immune response that
will protect a person from illness due to that agent. A therapeutic (treatment) vaccine is given
after infection and is intended to reduce or arrest disease progression. A. preventive
(prophylactic) vaccine is intended to prevent initial infection. Agents used in vaccines may be
whole-killed (inactive), live-attenuated (weakened) or artificially manufactured. A vaccine may
further comprise a diluent, an adjuvant, a carrier, or combinations thereof, as would be readily
understood by those in the art.
[052] A vaccine may be comprised of separate components. As used herein, "separate
components" refers to a situation wherein the term vaccine actually comprises two discrete
vaccines to be administered separately to a subject. In that sense, a vaccine-comprised of
separate components may be viewed as a kit or a package comprising separate vaccine
components. For example, in the context of the instant Invention, a package may comprise an
attenuated sporozoite component and recombinant subunit vaccine component, including but not
limited to a necombinant protein, recombinant virus, recombinant bacteria, recombinant parasite,
DNA vaccine, or RNA vaccine.

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[053] An "effective" immunizing dosage may range between 1000 and 10 million
sporozoites, but could be lower if the immunogenicity/potency of the vaccine is increased. The
vaccine may be administered on multiple occasions. An "effective" number of inoculations may
range between 1 and 6 doses within a year, and "booster" doses in subsequent years.
[054] Both the foregoing general description and the following detailed description are
exemplary and explanatory only and are not restrictive of the invention, as claimed. Moreover,
the invention is not limited to the particular embodiments described, as such may, of course,
vary. Further, the terminology used to describe particular embodiments is not intended to be
limiting, since the scope of the present invention will be limited only by its claims.
[055] With respect to ranges of values, the invention encompasses each intervening value
between the upper and lower limits of the range to at least a tenth, of tenth lower limit's unit, unless
the context clearly indicates otherwise. Further, the invention encompasses any other stated
intervening values. Moreover, the invention also encompasses ranges excluding either or both of
the upper and lower limits of the range, unless specifically excluded from the stated range.
[056] Unless defined otherwise, the meanings of all technical and scientific terms used
herein are those commonly under stood by one of ordinary skill in the art to which this invention
belongs. One of ordinary skill in the art will also appreciate that any methods and materials
similar or equivalent to those described herein can also be used to practice or test the invention.
Further, all publications mentioned herein are incorporated by reference.
[057] It must be noted that, as used herein and in the appended claims, the singular forms
"a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Thus,
for example, reference to "an attenuated sporozoite vaccine" includes a plurality of such
sporozoites and reference to "the agent" includes reference to one or more agents and equivalents
thereof known to those skilled in the art, and so forth.
[058] Further, all numbers expressing quantities of ingredients, reaction conditions, %
purity, and so forth, used in the specification and claims, are modified by the term "about,"
unless otherwise indicated. Accordingly, the numerical parameters set forth in-the specification
and claims are approximations that may vary depending upon the desired properties of the
present invention. At the very least, and not as an attempt to limit the application of the doctrine
of equivalents to the scope of the claims, each numerical parameter should at least be construed
in light of the number of reported significant digits, applying ordinary rounding techniques.
Nonetheless, the numerical values set forth in the specific examples are reported as precisely as
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possible. Any numerical value, however, inherently contains certain errors from the standard
deviation of its experimental measurement.
[059] Obviously, many modifications and variations of the present invention are possible
in light of the above teachings. It is therefore to be understood that, within the scope of the
appended claims, the invention many be practiced otherwise than as specifically described,
[060] The following examples further illustrate the invention. They are merely
illustrative of the invention and disclose various beneficial properties of certain embodiments of
the invention. These examples should not be construed as limiting the invention.
EXAMPLES
EXAMPLE 1
Comparative Infectivity of Intradermal, Intramuscular, Subcutanneous and
Intravenous Injection of Sporozoites
[061 ] A study was conducted to investigate the comparative infectivity of freshly
dissected sporozoites delivered intradermally (ID), intramuscularly (IM), subcutaneously (SQ) or
intravenously (IV). It is noted that IV administration is considered to be the most reliable
methods for achieving infection.
[062] Methods: BALB/c mice were infected with Plasmodium yoelii sporozoites band-
dissected from salivary glands by ID, IM, SQ, or IV administration. The level of infection was
determined by assessing thick blood films from day 1 through day 14 after administration. The
results are shown in Table I.
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[063] These data demonstrate that it is possible to routinely infect BALB/c mice by
delivery of sporozoites in the skin, muscle, or subcutaneous tissue,
EXAMPLE 2
Comparative Infectivity of Multiple Dose of Sporozoites Administered Intradermally
Intramuscularly, Subcutaneously or Intravenously
[064] A study was conducted to investigate the comparative infectivity with lesser
numbers of freshly dissected sporozoites than used in Example 1.
[065 ] Methods: B ALB/c mice infected with Plasmadium yoelii sporozoites hand-
dissected from salivary glands by multiple routes [intradermal (ID), intramuscular (IM),
subcutaneous (SQ) or intravenous (IV)]. Infection was determined by assessing thick blood films
through day 14 after infection. The results are shown in Table II.
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[066] These data show that administration of small numbers of Plosmodium yoelii
sporozoites hand-dissected from salivary glands by the ID, IM, or SQ routes leads to infections
in mice with nearly the same efficiency as as by the IV route. Since we theorize that there is a
direct correlation/assocation between the infectivity of unirradiated sporozoites when
administered by a particular method, and the capacity of these sporozoites when irradiated and
delivered by that method to to elicit protective immunity, these data suggest that it should be
feasible to successfully immunize by the ID, IM, and SQ routes as well as by the standard IV
route.
EXAMPLE 3
Protective Efficacy of Single Dose of Irradiated Sporozoites Administered by the
Intradermal, Intramuscular, or Intraveous Routes
[067] A study was conducted to investigate the comparative protection provided by
immunization with a single dose of 150,000 radiation attenuated sporozoites.
[068] Method: BALB/c mice were inoculated with a single dose of 150,000 radiation
attenuated (10,000 Rads/cGy) P. yoelii sporozoites by the ID, IM, or IV routes. The sporozoites
for immunization were obtained by density gradient centrifugation. The inoculated mice were
challenged 10 days later by injection of 100 Plasmodium yoelii sporozoites hand-dissected from
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salivary glands. The infections were assessed through day 14 after challenge by thick blood
smear. The level of infection was evaluated on a scale of 1+ (barely detectable) to 4+ (heavy
infection,). The control group received no immunization inoculation. The results are shown in
Table III.
Table III
[069] These data demonstrate that administration of a single dose of irradiated sporozoites
by the ID and IM routes elicits a protective immune response that provides protection against
sporozoite challenge comparable to the protection seen after administration of a single dose of
irradiated sporozoites by the IV route. This finding was predicted by the infectivity demonstrated
in Examples 1 and 2 above. Inasmuch as IM and ID methods are more easily used with large
numbers of people and the administration can be carried out with much greater safety and ease
than by IV administration, the present invention makes possible the effective immunization of
significant populations with attenuated sporozoites in a manner more facile than heretofore
demonstrated. In fact it makes it possible to coaceive of for the first time a practical attenuated
sporozoite vaccine. Administration of the single dose of irradiated sporozoites led to a dramatic
reduction of parasite burden in the the mice that were challenged, an effect thought by many
malaria vaccinologists to potentially be adequate to significantly reduce morbidity and mortality
of malaria in recipients. However, It did not completely protect against infection.
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EXAMPLE 4
Protective Efficacy of Three Doses of Irradiated Sporozoites Administered by the
Subcutaneous or Intraveous Routes
[070] A study was conducted to investigate the comparative protection provided by
immunization with a standard regimen of three doses of radiation attenuated Plasmodium yoelli
sporozoites by the ID or IV routes; a regimen expected to elicit complete protection against
sparozoite challenge.
[071] Method: BALB/c mice were inoculated with a first dose of 50,000 radiation
attenuated (10,000 RADS/cGy) Plasmodium yoelii sporozoites by the SQ or IV routes. The mice
received two booster doses of 30,000 irradiated sporozoites (total of 110,000 sporozoites divided
into 3 doses). The sporozoites for immunization were obtained by density gradient
centrifugation. The inoculated mice were challenged 14 days after last booster dose with 100
Plasmodium yoelii sporozoites hand-dissected from salivary glands. The infections were
assessed through day 14 after challenge by thick blood smear. Infection was assessed as present
or absent The results are shown in Table IV.
[072] The data in Table IV clearly demonstrate that one can achieve 100% protection
against infection by subcutaneous administration of sporozoites (SQ). These results were
predicted by the results of studies shown in Example 1, Example 2, and Example 3, but for the
first time ever demonstrated in this experiment. Given the comparability in infectivity by the
SQ, ID, and IM routes (Example 2), it seems obvious that administration of sporozoites by those
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routes would provide comparable protection. The 100% protection reported in Example 4 stands
in stark contrast to the 0% protection with subcutaneous immunization of A/J mice with radiation
attenuated P, berghei sporozoites reported previously [21]. As stated above we believe that our
discovery was made possible by our recognition that the P. yoelii-BALB/c model is more
relevant to P. falciparum in humans, than is the P. berghei-A/J mouse model system.
EXAMPLE 5
Infectivitv of Sporozoites Isolated by Density Gradient Centrifugation as Compared
to by Hand Dissection of Salivary Glands When Administered by the intravenous Route
[073] In EXAMPLES 3 and 4 the mice were immunized by administration of irradiated
sporozoites that had been isolated by density gradient centrifugation. It had been our assumption
that sporozoites isolated by density gradient centrifugation of the head and thorax of the
mosquitoes are less infective than are sporozoites hand-dissected from salivary glands. If that is
the case, and there is a direct association between the infectivity of sporozoites and their capacity
to elicit protective immunity as stated above (paragraph [068]), then it should require far fewer
sporozoites hand-dissected from salivary glands than sporozoites isolated by density gradient
centrifugation to achieve protective immunity. The inventors therefore first conducted an
experiment comparing the infectivity of P. yoelii sporozoites isolated by density gradient
centrifugation to those isolated by hand dissection of salivary glands .
[074] Method: P. yoelii sporozoites were isolated from Anopheles Stephensl mosquitoes
by density gradient centrifugation or by hand dissection of salivary glands. BALB/c mice were
inoculated by intravenous injection with differing numbers of sporozoites. The infections were
assessed through day 14 after challenge by thick blood smear. Infection was assesed as present
or absent The results are shown in Table V.
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50% Infection Dose (IP 50) 4.9 433
[075] The data in Table V clearly demonstrate that sporozoites hand-dissected from
salivary glands are more infective than are sporozoites isolated by density gradient
centrifugation. The 50% infectious dose is more than 80 times greater for sporozoites isolated by
density gradient centrifugation. If the hypothesis is correct that the protective efficacy of a lot of
attenuated sporozoites is directly associated with the infectivity of the lot of sporozoites before
they were attenuated, then these data would indicate that the numbers of attenuated sporozoites
required to achieve protection would be substantially less for sporozoites isolated by hand,
dissection of salivary glands sas compared to sporozoites isolated by density gradient
centrifugation which has been the standard way of isolating sporozoites for immunization,
studies in the P. yoelii-BALB/c model system.
EXAMPLE 6
Protective Efficacy of Sporozoites Isolated by Density Gradient Centrifugation as
Compared to by Hand Dissection When Administered by the Intravenous Route
[076] Based on the results of the infectivity experiment in EXAMPLE 5, a protective
efficacy experiment was designed. The protective efficacy of a regimen of irradiated sporozoites
isolated by density gradient centrifugation which was known based on previous-experience to
give 90% protection, was compared to the capacity of much lower doses of irradiated sporozoites
isolated by hand dissection of salivary glands to achieve protective immunity.
[077] Method: Anopheles stephensi mosquitoes infected with P. yoelii sporozoites were
irradiated with 10,000 Rads/cGy. Sporozoites were isolated by density gradient centrifugation or
by band dissection of salivary glands. BALB/c mice were inoculated by intravenous injection of
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three doses of irradiated P. yoelii sporozoites at 2 week intervals. Group 1 received irradiated
sporozoites isolated by density gradient centrifugation (24,000, 8,000, and 8,000 for first,
second, and third doses respectively). Groups 2-5 received sporozoites isolated by hand
dissection of salivary glands, Group 6 received no immunizations. The mice in Groups 1-6 were
challenged with 100 P. yoelii sporozoites isolated by hand-dissection of salivary glands 14 days
after the third immunizing dose. The infections were assessed through day 14 after challenge by
thick blood smear. Infection was assessed as present or absent. The results are shown in Table
VI.
[078] The data in Table VI demonstrate that mice immunized with a total of 40,000
irradiated P. yoelii sporozoites (24000, 8000, 8000) isolated by density gradient centrifugation
had 90% protection. Mice immunized with a total of 7500 irradiated sporozoites (4500, 1500,
1500) isolated by hand dissection of salivary glands had 100% protection. These data, when
taken with the data in EXAMPLE 5 indicate that there is a direct association between the
inactivity of a preparation of sporozoites, and the protective efficacy they can elicit. In fact it is
not yet clear how low one can go in terms of doses of irradiated, hand-dissected sporozoites, and
still achieve 90%-100% protective efficacy. These data indicate that immunizing with small
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doses of irradiated sporozoites, whether by the IV, ID, IM, or SQ routes, will lead to protective
efficacy.
[079] These data also support the hypothesis that the P. yoelii-BALB/c model system
more closely predicts what occurs in humans with P. falciparum than does the P. berghei-A/J
mouse model system, in part because of the much higher infectivity of sporozoites in the P.
yoelii system. Humans can be fully immunized by the bite of 1000 irradiated, P. falciparum
infected mosquitoes [34]. It is thought that a mosquito inoculates no more than 10 sporozoites
when it feeds [51]. If that is the case, then fully immunized and protected humans are probably
inoculated with only 10,000 sporozoites [50]. In contrast, in the P. berghei-A/J mouse model
system greater than 100,000 sporozoites isolated from hand-dissected salivary glands were used
to achieve protection by intravenous administration, and this immunizing dosage regimen
provided no protection when administered subcutaneously [21]. In Example 6 it is demonstrated
that administration to BALB/c mice of 750G P. yoelii sporozoites isolated by hand dissection of
salivary glands provided 100% protection. The fact that BALB/c mice immunized with
attenuated P. yoelii sporozoites and humans immunized with attenuated P. falclpurum
sporozoites are protected after exposure to similar numbers of attenuated sporozoites, and A/J
mice immunized, with P. berghei sporozoites are immunized with more than 10 times the
quantity of sporozoites, supports our hypothesis that the P. yoelii-BALB/c model will be more
predictive of what will occur in humans than the P. berghei-A/J model system.
CONCLUSIONS
[080] The process of developing an effective, sustainable vaccine against infections like
P. falciparum has proven to be slower, more difficult and complex than expected. There is no
licensed malaria vaccine, but it is now known that immunization with radiation attenuated P .
falciparum sporozoites by the bite of greater than a 1000 infected mosquitoes provides sterile
protective immunity in greater than 90% of immunized individuals for at least 10.5 months
against multiple isolates of P. falciparum from throughout the world. One of the major obstacles
to making this immunization regimen into a vaccine for humans has been the fact that it is not
possible to provide a regulated vaccine to large numbers of individuals by the bite of infected
mosquitoes. Furthermore, work by a number of scientists indicated that excellent protection
could only be achieved in the mouse model system by intravenous administration of attenuated
sporozoites, a method of administration that is not in general used for vaccination, because it is
technically difficult and potentially more dangerous than are standard methods of administration.
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Because methods of administration conventionally used in humans for immunization like
subcutaneous and intramuscular inoculation did not lead to adequate protective immunity in this
mouse model system, it was heretofore not considered possible to develop an attenuated
sporozoite vaccine for humans. Utilizing a different model system than that used by previous
investigators, we have discovered a method of administering sporozoites that leads to high level
protection and is practical, safe, and accepted. This discovery should facilitate utilization of this
method of administering attenuated sporozoites to develop and provide a practical, mass-
delivered attenuated sporozoite malaria vaccine.
[081] The following publications as well as those mentioned anywhere else in this
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41. Doolan DL, Southwood S, Chesnut R, Appella E, Gomez E, Richards A,
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29

WO 2004/045559 PCT/US2003/037498
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[082] Other embodiments of the invention will be apparent to those skilled in the art from
consideration of the specification and practice of the invention disclosed herein. It is intended
that the specification and examples be considered as exemplary only, with a true scope and spirit
of the invention being indicated by the following claims.
30

We claim:
1. A pharmaceutical composition for stimulating an immune response
in mammalian and human hosts by parenteral, non-intravenous inoculation, said
composition comprising metabolically active, attenuated Plasmodiunt sporozoite
parasites and a carrier.
2. The pharmaceutical composition of Claim 1 wherein said
sporozoites are obtained from hand-dissected Anopheles mosquito salivary
glands.
3. The pharmaceutical composition of Claim 1 wherein the species of
said Plasmodium parasite is falciparum .
4. The pharmaceutical composition of Claim 1 comprising
Plasmodium falciparum sporpzoites and at least one additional species of
Plasmodium sporozoite.
5. The pharmaceutical composition of Claim 1 wherein said attenuated
sporozoite parasites invade cells of said host.
6. The pharmaceutical composition of Claim 5 whenein said cells
comprise hepatic cells and said parasites do not induce subsequent hepatic cell
rupture.
7. The pharmaceutical composition of Claim 5 wherein said cells
comprise hepatic cells, said parasites induce hepatic cell rupture, and said
parasites are not capable of subsequent development within host erythrocites.
8. The pharmaceutical composition of Claim 1 wherein attenuation is
achieved by a means for gene alteration.
9. The pharmaceutical composition of Claim 8 wherein said alteration
means is chosen from a group consisting of irradiation genetic manipulation,
and treatment of sporozoites with chemicals.
10. The pharmaceutical composition of Claim 9 comprising radiation-
attenuated Plasmodium sporozoites.
31



11. The pharmaceutical composition of Claim 10 wherein dosage of
attenuating radiation is at least 12 ,000 eGy and no more than 23,000 cGy.
12. The pharmaceutical composition of Claim 11 wherein dosage is
proximate to 15,000 cGy.
13. The pharmaceutical composition of. Claim 1 comprising at least
1000, but not more than 10,000,000, sporozoites.
14. The pharmaceutical composition of Claim 13 comprising at least
5,000,0 but not more than 100, 000, sporozoites.

15. The pharmaceutical composition of Claim 14 comprising at least
10,000, but not more than 50,000, sporozoites.
16. The pharmaceutical composition of Claim 1 wherein administration
of said composition to a mammalian or human host prevents malaria-specific
pathology in said host after subsequent introduction into said host of infections
Plasmodium sporozoites.
17. A pharmaceutical vaccinatiou kit for stimulating an immune
response in mammalian and human hosts, said kit comprising a pharmaceutical
composition of metabolically active, attenuated Plasmodium sporozoite
parasites, a carrier, and means for parenteral non-intravenous inoculation.
18. The vaccination kit of Claim 17 wherein said inoculation means is a
needle.
19. The vaccination kit of Claim 17 wherein said inoculation means is a
micro-needle array.
: 20. The vaccination kit of Claim 17 wherein said inoculation means is a
needle-free ballistic injector.
21. The vaccination kit of Claim 17 wherein said inoculation means is a
needle-free particle injector.
22. The vaccination kit of Claim 17 wherein the species of said
Plasmodium sporozoites comprises falciparum.
32 . -

23. The vaccination kit of Claim 17 wherein said attenuated sporozoite
parasites invade cells of said host.
24. The vaccination kit of Claim 17 wherein attenuation is achieved by a
means for gene alteration.
25. The vaccination kit of Claim 24 wherein said alteration means is
chosen from a group consisting of irradiation, genetic manipulation; and
treatment of sporozoites with chemicals.
26. The vaccimation kit of Claim 25 comprising radittion-attenuated
Plasmodium sporozoites.
27. The vaccination kit of Claim 17 comprising at least 1000, but not
more than 10,000,000, sporozoites.
28. The vaccination kit of Claim 27 comprising at least 5,000, but not
more than 100,000, sporozoites.
29. The vaccination kit of Claim 28 comprising at least 10,000, but no
more than 50,000, sporozoites.
30. The vaccination kit of Claim 17 wherein administration of said
composition by said inoculation means, to a mammalian or human host, prevents
malaria-specific pathology in said host, after subsequent introduction, into said .
host of infectious Plasmodium sporozoites. 33

The invention comprises a novel method for protecting subjects against malaria. The method of the invention invoices
inoculation with attenuated sporozoites. and in particular, but not limited 10 subcutaneous, intramuscular, intradermal, mucosal,
submucosal, and cutaneous administration.


Documents:


Patent Number 217420
Indian Patent Application Number 01170/KOLNP/2005
PG Journal Number 13/2008
Publication Date 28-Mar-2008
Grant Date 26-Mar-2008
Date of Filing 17-Jun-2005
Name of Patentee SANARIA INC.
Applicant Address 12115 PARKLAWN DRIVE, ROCKVILLE, MD 20852, U.S.A.
Inventors:
# Inventor's Name Inventor's Address
1 HOFFMAN, STEPHEN, L 308 ARGOSY DRIVE, GAITHERSBURG, MD 20878, U.S.A.
2 LUKE, THOMAS, C 4101 BROOKEVILLE ROAD, BROOK VILLE, MD 20833, U.S.A.
PCT International Classification Number G01N 33/536
PCT International Application Number PCT/US2003/037498
PCT International Filing date 2003-11-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/427,911 2002-11-20 U.S.A.