U.S. patent application number 13/470128 was filed with the patent office on 2012-11-15 for pharmaceutical compositions comprising attenuated plasmodium sporozoites and glycolipid adjuvants.
Invention is credited to Sumana CHAKRAVARTY, Stephen L. HOFFMAN, Moriya TSUJI.
Application Number | 20120288525 13/470128 |
Document ID | / |
Family ID | 47142026 |
Filed Date | 2012-11-15 |
United States Patent
Application |
20120288525 |
Kind Code |
A1 |
CHAKRAVARTY; Sumana ; et
al. |
November 15, 2012 |
PHARMACEUTICAL COMPOSITIONS COMPRISING ATTENUATED PLASMODIUM
SPOROZOITES AND GLYCOLIPID ADJUVANTS
Abstract
Disclosed herein are pharmaceutical compositions comprising
Plasmodium sporozoite-stage parasites and compatible glycolipid
adjuvants useful in vaccines for preventing or reducing the risk of
malaria. In particular, human host range Plasmodium and analogues
of .alpha.-galactosylceramide (.alpha.-GalCer), a ligand for
natural killer T (NKT) cells, are combined in pharmaceutical
compositions, which are useful as vaccines against malaria. Methods
of use are also provided.
Inventors: |
CHAKRAVARTY; Sumana;
(Derwood, MD) ; HOFFMAN; Stephen L.;
(Gaithersburg, MD) ; TSUJI; Moriya; (New York,
NY) |
Family ID: |
47142026 |
Appl. No.: |
13/470128 |
Filed: |
May 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61485092 |
May 11, 2011 |
|
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Current U.S.
Class: |
424/272.1 |
Current CPC
Class: |
A61K 2039/55511
20130101; A61K 39/015 20130101; A61K 2039/522 20130101; A61P 33/06
20180101; A61K 9/0019 20130101; A61K 39/39 20130101; Y02A 50/412
20180101; A61K 2039/54 20130101; A61K 9/0021 20130101 |
Class at
Publication: |
424/272.1 |
International
Class: |
A61K 39/015 20060101
A61K039/015; A61P 33/06 20060101 A61P033/06 |
Claims
1. A pharmaceutical composition comprising one or more species of
live Plasmodium sporozoite-stage parasites, an excipient, and a
glycolipid adjuvant, wherein said adjuvant is represented by the
structure of formula 1; ##STR00007## wherein R is selected from the
group consisting of: R.dbd.(CH.sub.2).sub.5Ph(p-OMe);
R.dbd.(CH.sub.2).sub.7Ph(p-OMe); R.dbd.(CH.sub.2).sub.7Ph(p-F);
R.dbd.(CH.sub.2).sub.10Ph(p-F); and
R.dbd.(CH.sub.2).sub.10Ph(p-CF.sub.3).
2. The pharmaceutical composition of claim 1 wherein
R.dbd.(CH.sub.2).sub.10Ph(p-F).
3. The pharmaceutical composition of claim 1 wherein said species
are selected from the group consisting of: P. falciparum, P. vivax,
P. ovale, P. knowlesi, P. malariae, and P. yoelii.
4. The pharmaceutical composition of claim 3 wherein said species
comprises P. falciparum.
5. The pharmaceutical composition of claim 3 wherein said
sporozoite-stage parasites are attenuated.
6. A method of reducing the risk of malaria in a host exposed to
pathogenic Plasmodium species parasites, said method comprising
administration of one or more doses of a pharmaceutical composition
to said host prior to said exposure, wherein said pharmaceutical
composition comprises one or more species of live attenuated
Plasmodium sporozoite-stage parasites and an excipient; and wherein
a glycolipid adjuvant represented by the structure of formula 1 is
co-administered; ##STR00008## wherein R is selected from the group
consisting of: R.dbd.(CH.sub.2).sub.5Ph(p-OMe);
R.dbd.(CH.sub.2).sub.7Ph(p-OMe); R.dbd.(CH.sub.2).sub.7Ph(p-F);
R.dbd.(CH.sub.2).sub.10Ph(p-F);
R.dbd.(CH.sub.2).sub.10Ph(p-CF.sub.3).
7. The method of claim 6 wherein
R.dbd.(CH.sub.2).sub.10Ph(p-F).
8. The method of claim 6 wherein said host is a mammalian host.
9. The method of claim 8 wherein said host is a human host.
10. The method of claim 6 wherein said one or more doses comprise
no more than 150,000 sporozoites.
11. The method of claim 10 wherein said one or more doses comprise
no more than 50,000 sporozoites.
12. The method of claim 11 wherein said one or more doses comprise
no more than 25,000 sporozoites.
13. The method of claim 6 wherein the number of doses is no more
than 3.
14. The method of claim 13 wherein the number of doses is no more
than 2.
15. The method of claim 14 wherein the number of doses is no more
than 1.
16. The method of claim 6 wherein said Plasmodium species of said
pharmaceutical composition are selected from the group consisting
of: P. falciparum, P. vivax, P. ovale, P. knowlesi, and P.
malariae.
17. The method of claim 16 wherein said Plasmodium species of said
pharmaceutical composition comprises P. falciparum.
18. The method of claim 6 wherein said pharmaceutical composition
is administered by a parenteral route chosen from the group
consisting of intravenous, intramuscular, intradermal, and
subcutaneous.
19. A malaria vaccine comprising one or more species of live
Plasmodium sporozoite-stage parasites, an excipient, and a
glycolipid adjuvant, wherein said adjuvant is represented by the
structure of formula 1; ##STR00009## wherein R is selected from the
group consisting of: R.dbd.(CH.sub.2).sub.5Ph(p-OMe);
R.dbd.(CH.sub.2).sub.7Ph(p-OMe); R.dbd.(CH.sub.2).sub.7Ph(p-F);
R.dbd.(CH.sub.2).sub.10Ph(p-F); and
R.dbd.(CH.sub.2).sub.10Ph(p-CF3).
20. The vaccine of claim 19 wherein
R.dbd.(CH.sub.2).sub.10Ph(p-F).
21. The vaccine of claim 19 wherein said species are selected from
the group consisting of: P. falciparum, P. vivax, P. ovale, P.
knowlesi, P. malariae, and P. yoelii.
22. The vaccine of claim 21 wherein said species comprises P.
falciparum.
23. The vaccine of claim 19 wherein said sporozoite-stage parasites
are attenuated.
24. A method of reducing the risk of malaria in a host exposed to
pathogenic Plasmodium species parasites, said method comprising
administration of one or more doses of a pharmaceutical composition
to said host prior to said exposure, wherein said pharmaceutical
composition comprises one or more species of live Plasmodium
sporozoite-stage parasites and an excipient; and, wherein a
glycolipid adjuvant is co-administered, wherein said adjuvant is
represented by the structure of formula 1; ##STR00010## wherein R
is selected from the group consisting of:
R.dbd.(CH.sub.2).sub.5Ph(p-OMe); R.dbd.(CH.sub.2).sub.7Ph(p-OMe);
R.dbd.(CH.sub.2).sub.7Ph(p-F); R.dbd.(CH.sub.2).sub.10Ph(p-P);
R.dbd.(CH.sub.2).sub.10Ph(p-CF.sub.3); and wherein an antimalarial
drug was previously administered such that the concentration of
said drug in the bloodstream of said host is sufficient to prevent
the clinical manifestations of malaria.
25. The method of claim 24 wherein
R.dbd.(CH.sub.2).sub.10Ph(p-F).
26. The method of claim 24 wherein said host is a mammalian
host.
27. The method of claim 26 wherein said host is a human host.
28. The method of claim 24 wherein said antimalarial drug is
chloroquine.
29. The method of claim 24 wherein said one or more doses comprise
no more than 150,000 sporozoites.
30. The method of claim 29 wherein said one or more doses comprise
no more than 50,000 sporozoites.
31. The method of claim 30 wherein said one or more doses comprise
no more than 25,000 sporozoites.
32. The method of claim 24 wherein the number of doses is no more
than 3.
33. The method of claim 32 wherein the number of doses is no more
than 2.
34. The method of claim 33 wherein the number of doses is no more
than 1.
35. The method of claim 24 wherein said Plasmodium species of said
pharmaceutical composition are selected from the group consisting
of: P. falciparum, P. vivax, P. ovate, P. knowlesi, and P.
malariae.
36. The method of claim 35 wherein said Plasmodium species of said
pharmaceutical composition comprises P. falciparum.
37. The method of claim 24 wherein said pharmaceutical composition
is administered by a parenteral route chosen from the group
consisting of intravenous, intramuscular, intradermal, and
subcutaneous.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from the U.S. Provisional
application 61/485,092, filed May 11, 2011, the contents of which
are hereby incorporated by reference their entireties.
FIELD OF THE INVENTION
[0002] The invention disclosed herein relates generally to
pharmaceutical compositions comprising Plasmodium parasites useful
as immunogens in vaccines for preventing or reducing the risk of
malaria. More particularly, the invention relates to pharmaceutical
compositions comprising Plasmodium sporozoite-stage parasites and
glycolipid adjuvants wherein the compositions are useful for
eliciting an immune response, conferring protective immunity in a
host so as to prevent malaria, and reducing the incidence of
malaria in hosts subsequently challenged with pathogenic
Plasmodium, more particularly in mammalian or human hosts, and the
invention relates to vaccines and methods of using the provided
pharmaceutical compositions in vaccines for the prevention of
malaria.
BACKGROUND OF THE INVENTION
[0003] There are >200 million malaria cases and .about.1 million
deaths per year caused by Plasmodium falciparum (Pf) (World Health
Organization. Global Malaria Programme. World Malaria Report 2010:
World Health Organization, 2010; Murray C J, et al. Global malaria
mortality between 1980 and 2010: a systematic analysis. 2012 Lancet
379: 413-431). Recently, progress has been made controlling malaria
due to investment of billions of dollars in the use of bednets,
insecticides, and drugs. However, as highlighted in a recent
editorial (Editorial: Malaria 2010: More Ambition and
Accountability Please 2010 Lancet 375:1407), a commercially
available malaria vaccine is still badly needed. To prevent
infection, disease, and transmission an ideal single stage vaccine
should target the pre-erythrocytic (sporozoite and liver) stages
(Plowe C. V. et al. The potential role of vaccines in the
elimination of falciparum malaria and the eventual eradication of
malaria 2009 J. Infect. Dis. 200:1646-1649; Alonso P. L. et al. A
research agenda for malaria eradication: vaccines. 2011 PLoS Med 8:
e1000398). Such a vaccine would have huge public- and
private-sector markets. In public-sector markets it would be used
in infants, young children, and adolescent females (preventing
malaria during pregnancy) and for entire populations for
geographically focused malaria elimination campaigns (Id.).
Individuals from non-malarious countries who spend time in areas
with malaria (travelers, military, government officials, students,
business people, etc.) and middle and upper class residents of
countries with malaria comprise the private-sector market.
[0004] Data indicating a highly effective vaccine might be possible
came from trials in which volunteers immunized by the bites of
mosquitoes infected with radiation-attenuated Pf sporozoites had
high-level (>90%), sustained (.gtoreq.10 months) protection
against experimental challenge (Hoffman, S. L., et al. 2002 J. Inf.
Dis. 185:1155-64).
[0005] It has been shown that a vaccine incorporating live
attenuated Pf sporozoites can be manufactured (SANARIA.TM. PfSPZ
Vaccine). This process has recently been described (Hoffman S. L.
et al. Development of a metabolically active, non-replicating
sporozoite vaccine to prevent Plasmodium falciparum malaria. 2010
Hum. Vac. 6:97-106. DOI: 10396 [pii].). The ability of PfSPZ
Vaccine to induce antigen-specific immune responses in humans was
also demonstrated (Epstein, J. E., et al. Live Attenuated Malaria
Vaccine Designed to Protect Through Hepatic CD8.sup.+T Cell
Immunity 2011 Science 334 (6055):475-480).
[0006] It is thought that an attenuated PfSPZ vaccine delivered by
a parenteral non-intravenous route and capable of demonstrating a
protective efficacy comparable to that achieved with PfSPZ
administered IV would require large numbers of sporozoites and a
multi-dose regimen. In a mouse model, present data suggests that
approximately 7 times as many Plasmodium yoelii (Py) sporozoites
administered intradermal (ID) or subcutaneous (SC) are required
compared to IV administration in order to achieve >80%
protection in mice (Epstein, et al., Id.). A more promising
approach for the development of a highly effective parenteral
non-IV vaccine would likely include the use of an adjuvant.
[0007] The Adjuvant:
[0008] A glycolipid adjuvant that stimulates natural killer T-cells
(NKT) was identified in mice. (Gonzalez-Aseguinolaza G, et al.
Natural killer T cell ligand .alpha.-galactosylceramide enhances
protective immunity induced by malaria vaccines 2002 J. Exp. Med.
195: 617-624; U.S. Pat. No. 7,534,434). Using a single
IV-administered dose of radiation attenuated P. yoelii sporozoites
(suboptimal for protection) it was demonstrated in the
Gonzalez-Aseguiniola paper that distal intraperitoneal (IP)
administration of .alpha.-galactosylceramide (.alpha.-GalCer), a
ligand for natural killer T (NKT) cells, could induce a higher
degree of protection (>90%), than IV administration of
irradiated P. yoelii sporozoites alone, which conferred only 20%
protection.
[0009] Natural Killer T (NKT) cells are a subset of T cells that
co-express receptors of T cell and NK cell lineages and recognize
their cognate antigen presented by the MHC-like CD on antigen
presenting cells (APCs). The major subset of NKT cells are
distinguished by their restricted expression of an invariant TCR
(invTCR) and are termed iNKT cells. The increased potency of IV
administered sporozoites and distally administered IP adjuvant
described in Gonzalez-Aseguinolaza et al. correlated with enhanced
IFN-gamma secretion by CD8.sup.+ T cells and was dependent on iNKT
cells and CD1d (Id.). This first-identified iNKT TCR ligand,
.alpha.-GalCer, extracted from the Agelas mauritianus sea sponge,
was discovered while screening for compounds with anti-tumor
activity. It has a high affinity for CD1d, is a potent activator of
iNKT cells in both mouse and human, and has been used extensively
to study the function of iNKT cells (Brossay L, et al.
CD1d-Mediated Recognition of an A-Galactosylceramide by Natural
Killer T Cells is Highly Conserved through Mammalian Evolution 1998
J. Exp. Med. 188:1521-1528; Kobayashi E, et al. KRN7000, A Novel
Immunomodulator, and its Antitumor Activities 1995 Oncol. Res. 7:
529-534; Kawano T, et al., CD1d-restricted and TCR-mediated
activation of va14 NKT cells by glycosylceramides 1997 Science 278:
1626-1629. In vivo administration of .alpha.-GalCer in mice results
in a cascade of events beginning with signaling through the invTCR
by APCs expressing CD1d. Macrophages, dendritic cells, B cells,
Kupffer cells in the liver, and hepatocytes all have constitutive
expression of CD1d (Mandal M, et al., Tissue distribution,
regulation and intracellular localization of murine CD1 molecules
1998 Mol. Immunol. 35: 525-536; Brossay L., et al., Mouse CD1 is
mainly expressed on hemopoietic-derived cells 1997 J. Immunol. 159:
1216-1224; Roark, J. H., et al., A. CD1.1 expression by mouse
antigen presenting cells and marginal zone B cells 1998 J. Immunol.
160: 3121-3127).
[0010] Stimulated iNKT cells rapidly secrete pre-stored cytokines
(unlike traditional T cells) that reciprocally activate APCs
(Tomura M., et al., A novel function of Va14+CD4+NKT cells:
stimulation of IL-12 production by antigen presenting cells in the
innate immune system 1999 J. Immunol. 163: 93-101; Fujii, S., et
al., Activation of natural killer T cells by
.alpha.-galactosylceramide rapidly induces the full maturation of
dendritic cells in vivo and thereby acts as an adjuvant for
combined CD4 and CD8 T cell immunity to a coadministered protein
2003 J. Exp. Med. 198: 267-279) enhancing their ability to prime
CD4.sup.+ and CD8.sup.+ T cells (Fujii, S., et al., The linkage of
innate to adaptive immunity via maturing dendritic cells in vivo
requires CD40 ligation in addition to antigen presentation and
CD80/86 costimulation 2004 J. Exp. Med. 199: 1607-1618; Hermans, I.
F., et al., NKT cells enhance CD4+ and CD8+ T cell responses to
soluble antigen in vivo through direct interaction with dendritic
cells 2003 J. Immunol. 171: 5140-5147) to generate a powerful
cell-mediated immune response. In the paper of
Gonzales-Aseguinolaza et al., supra, the legend of FIG. 2A states
that a group of BALB/c mice was immunized subcutaneously with
irradiated sporozoites [P. yoelii] together with or without
administration of .alpha.-GalCer by the same route, and when
splenic lymphocytes were isolated and the number of
IFN-.gamma.-secreting CS-specific CD8.sup.+ and CD4.sup.+ T-cells
were determined by ELISPOT assay it was found that
co-administration of .alpha.-GalCer increased the number of
IFN-.gamma.-secreting CS-specific CD8.sup.+ cells seven fold. Thus,
the overall amplification of the adaptive immune response by iNKT
cells made them very attractive adjuvant targets.
[0011] Subsequently, .alpha.-GalCer has been demonstrated to have
adjuvant properties for influenza, HIV, and tumor vaccines in mice
(Huang, Y., et al., Enhancement of HIV DNA vaccine immunogenicity
by the NKT cell ligand, .alpha.-galactosylceramide 2008 Vaccine
26:1807-1816; Ko, S. Y., et al., .alpha.-Galactosylceramide can act
as a nasal vaccine adjuvant inducing protective immune responses
against viral infection and tumor 2005 J. Immunol. 175: 3309-3317;
Seino, K., et al., Natural killer T cell-mediated antitumor immune
responses and their clinical applications 2006 Cancer Sci. 97:
807-812).
[0012] Furthermore, because .alpha.-GalCer was discovered while
screening for compounds with anti-tumor properties, it has been
used in several clinical trials in cancer patients. Delivery by
pre-loading autologous PBMCs with .alpha.-GalCer in vitro or by
direct injection, .alpha.-GalCer was shown to be safe and well
tolerated. However, although modest enhancement of immune responses
was generally seen, its beneficial effects were limited (Giaccone,
G., et al., A phase I study of the natural killer T-cell ligand
.alpha.-galactosylceramide (KRN7000) in patients with solid tumors
2002 Clin. Cancer Res. 8:3702-3709; Ishikawa, A., et al., A phase I
study of .alpha.-galactosylceramide (KRN7000)-pulsed dendritic
cells in patients with advanced and recurrent non-small cell lung
cancer 2005 Clin. Cancer Res. 11: 1910-1917; Nieda, M., et al.,
Therapeutic activation of Va24+Vbeta11+ NKT cells in human subjects
results in highly coordinated secondary activation of acquired and
innate immunity 2004 Blood 103: 383-389).
[0013] Consequently, Tsuji and colleagues made an effort to find
analogues of .alpha.-GalCer with increased CD1d-binding and
iNKT-stimulatory properties. The particular advantages of
identifying a new glycolipid adjuvant similar to .alpha.-GalCer and
based on a CD1d-binding, iNKT-stimulatory effect are multi-fold.
First, the phenotype and functional properties of the CD1d
molecules and invTCR of iNKT cells have been conserved between
humans and mice, thereby allowing prediction of the activity of
related glycolipids in humans through mouse studies. Second,
.alpha.-GalCer itself has been approved and well characterized in
terms of safety and activity in humans. Third, related glycolipids
used as vaccine adjuvant could be administered in much smaller
quantities using a local parenteral route of administration (e.g.
intramuscular) than the larger doses of .alpha.-GalCer currently
dispensed IV for cancer therapy, thereby further minimizing
potential systemic side effects.
[0014] Tsuji and colleagues screened a library of synthetic
.alpha.-GalCer analogues and identified glycolipids with far
greater CD1d binding and activation of iNKT cells (Li, X., et al.,
Design of a potent CD1d-binding NKT cell ligand as a vaccine
adjuvant 2010 Proc Natl Acad Sci USA 107: 13010-13015; U.S. Pat.
No. 7,923,013). One such glycolipid, was 7DW8-5 (FIG. 1).
Structurally, 7DW8-5 possesses a fluorinated benzene ring at the
end of C10 length fatty acyl chain. It was selected due to its
superior ability to elicit cytokine production from human and mouse
iNKT cells and its adjuvant properties in mice when used in
combination with a suboptimal dose of a recombinant adenovirus
expressing P. yoelii CS protein. The adjuvant was co-administered
intramuscularly (IM) with the vaccine. The mice were challenged
with pathogenic P. yoelii sporozoites 2 weeks later. 7DW8-5
enhanced the malaria-specific CD8.sup.+ T cell response
significantly more than .alpha.-Gal Cer and also enhanced the
malaria-specific humoral response equally if not slightly stronger
than .alpha.-GalCer. Finally, 7DW8-5 was able to display a
significantly stronger adjuvant effect than .alpha.-GalCer in
enhancing protective efficacy of the adenovirus recombinant vaccine
after a single immunizing dose.
[0015] The practical considerations for the preclinical and
clinical development of 7DW8-5 as an adjuvant for candidate
recombinant subunit malaria vaccines was recently discussed (Padte,
N. N., et al., Clinical development of a novel CD1d-binding NKT
cell ligand as a vaccine adjuvant 2010 Clin. Immunol. doi:
10.1016/j.clim.2010.11.009).
[0016] There is a need for improved malaria vaccines. With regard
to malaria vaccines whose immunogen is live attenuated Plasmodium
parasites, particularly sporozoite-stage parasites, an adjuvant
that could reduce the numbers of doses and the dosages of each dose
required for highly effective protection would have enormous value
in the fight against malaria. For instance, a vaccine administered
in 1 or 2 doses would not only reduce the cost of goods to produce
it, but more importantly it would simplify the logistics of
delivery for travelers, for rapidly deployed military, or for
mass-immunization campaigns.
SUMMARY OF THE INVENTION
[0017] Disclosed herein are pharmaceutical compositions comprising
one or more species of live, Plasmodium sporozoite-stage parasites,
a glycolipid adjuvant, and an excipient.
[0018] Also disclosed are methods of using these pharmaceutical
compositions for reducing the risk of malaria in an individual and
reducing the incidence of malaria among a group of individuals
exposed to pathogenic Plasmodium parasites. These methods comprise
administration of 1 or more doses of the pharmaceutical
compositions provided herein prior to said exposure.
[0019] Also disclosed are improved malaria vaccines whose immunogen
comprises either live attenuated Plasmodium sporozoites, or live
non-attenuated sporozoites delivered along with the protection of
an anti-malarial drug such as chloroquine, each of which being
useful for the prevention of malaria. Compositions of live
Plasmodium sporozoites and the glycolipid adjuvants disclosed
herein are compatible in pharmaceutical compositions and
surprisingly effective in reducing the number of doses and/or
reducing the effective sporozoites dosage.
DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1--Depicts the structures of .alpha.-GalCer and
7DW8-5.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0021] The terms "about" or "approximately" means within one
standard deviation as per the practice in the art.
[0022] "Additive" as used herein as a noun is a compound or
composition added to a sporozoite preparation to facilitate
preservation of the preparation. Additives may include
cryoprotectants such as DMSO and glycerol, antioxidants, and the
like.
[0023] "Conferring protective immunity" as used herein refers to
providing to a population, a group of individuals or a host (i.e.,
an individual), the ability to generate an immune response which in
turn protects against, i.e. prevents, a disease, malaria in this
case, caused by a pathogen (e.g. Plasmodium falciparum) such that
the clinical manifestations, pathology, or symptoms of disease in a
host exposed to the pathogen are reduced as compared to a
non-treated host, or such that the rate at which infection, or
clinical manifestations, pathology, or symptoms of disease appear
within a population are reduced, as compared to a non-treated
population, group or individual.
[0024] "Immune response" as used herein means a response in the
recipient to the introduction of attenuated sporozoites generally
characterized by, but not limited to, production of antibodies
and/or T cells. Generally, an immune response may be a cellular
response such as induction or activation of CD4+ T cells or CD8+ T
cells specific for Plasmodium species epitopes, a humoral response
of increased production of Plasmodium-specific antibodies, or both
cellular and humoral responses. With regard to a malaria vaccine,
the immune response established by a vaccine comprising sporozoites
includes but is not limited to responses to proteins expressed by
extracellular sporozoites or other stages of the parasite after the
parasites have entered host cells, especially hepatocytes and
mononuclear cells such as dendritic cells and/or to components of
said parasites. In the instant invention, upon subsequent challenge
by infectious organisms, the immune response prevents or exhibits
development of pathogenic parasites to the asexual erythrocytic
stage that causes disease.
[0025] "Intravenous" (abbreviated as IV) as defined herein means
intentional introduction, directly into the lumen of an identified
large blood vessel such as a vein.
[0026] "Metabolically active" as used herein means alive, and
capable of performing sustentative functions and some life-cycle
processes. With regard to attenuated sporozoites this includes
sporozoites that are capable of invading hepatocytes in culture and
in vivo, progressing through some developmental stages within
hepatocytes, and displaying de novo expression of stage-specific
proteins.
[0027] "Parenteral" as defined herein means not through the
alimentary canal, but rather by introduction through some other
route, as intradermal (ID), subcutaneous (SC), intramuscular (IM),
intraperitoneal (IP), intravenous (IV), and the like.
[0028] "Pharmaceutical composition" as defined herein means a
sterile composition comprising one or more active ingredients,
produced aceptically, under cGMP protocol, such that the
composition would be acceptable for use in human subjects.
[0029] "Therapeutic" as defined herein relates to reduction of
clinical manifestations or pathology which have already become
manifest. A "therapeutically effective amount" as used herein means
an amount sufficient to reduce the clinical manifestations,
pathology, or symptoms of disease in an individual, or an amount
sufficient to decrease the rate at which clinical manifestations,
pathology, or symptoms of disease appear within a population.
[0030] "Vaccine" as used herein is a preparation comprising an
immunogenic agent and a pharmaceutically acceptable diluent in
combination with excipient, adjuvant and/or additive or protectant.
The immunogen may be comprised of a whole infectious agent or a
molecular subset of the infectious agent (produced by the
infectious agent, or produced synthetically or recombinantly). When
the vaccine is administered to a subject, the immunogen stimulates
an immune response such that, upon subsequent challenge with
infectious agent, will protect the subject from illness or mitigate
the pathology, symptoms or clinical manifestations caused by 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 or reduce the rate or burden of the infection. Agents
used in vaccines against a parasitic disease such as malaria may be
whole-killed (inactive) parasites, live-attenuated parasites
(unable to fully progress through their life cycle), live, fully
infectious parasites administered with an anti-malarial drug, or
purified or artificially manufactured molecules associated with the
parasite e.g. recombinant proteins, synthetic peptides, DNA
plasmids, and recombinant viruses or bacteria expressing Plasmodium
proteins. A vaccine may comprise sporozoites along with other
components such as excipient, diluent, carrier, anti-malarial drug,
preservative, adjuvant or other immune enhancer, or combinations
thereof, as would be readily understood by those in the art.
[0031] Chemical nomenclature as used herein is as follows: "R"
represents a variable chemical structure attached at the indicated
location in a family of compounds; "Me" represents a methyl group
attached at the indicated location; "Ph" represents a phenolic ring
attached at the indicated location; "(p-OMe)" or "(p-F)" or
"(p-CF.sub.3)" following Ph represents the corresponding moiety
located at the para position of the phenolic ring; "F" represents a
fluoride moiety located attached at the indicated location; "O"
represents an oxygen moiety, "C" represents a carbon moiety; and
"H" represents a hydrogen moiety.
Pharmaceutical Compositions Comprising Plasmodium Sporozoites
[0032] In addition to the live attenuated PfSPZ Vaccine described
above, there is another approach to whole sporozoite malaria
vaccine development called chemoprophylaxis with sporozoites (CPS),
and requires the bite of only 45 mosquitoes (3 times 15 mosquitoes)
carrying fully infectious PfSPZ of volunteers taking chloroquine
chemoprophylaxis to achieve 95% sterile protective immunity (19/20
volunteers) and that lasts for at least 28 months (Roestenberg M,
et al. Protection Against a Malaria Challenge by Sporozoite
Inoculation. 2009 N. Eng. J. Med. 361: 468-477; Roestenberg M, et
al. Long-term Protection Against Malaria After Experimental
Sporozoite Inoculation: an Open-label Follow-up Study. 2011 Lancet
377: 1770-1776). This is 20-25 times fewer PfSPZ-infected
mosquitoes than the 1,000 required for the irradiated PfSPZ
approach (Hoffman S L, et al. Protection of humans against malaria
by immunization with radiation-attenuated Plasmodium falciparum
sporozoites 2002 J. Infect. Dis. 185:1155-1164). As disclosed in
Roestenberg 2009, incorporated herein by reference, the
antimalarial drug (e.g., chloroquine) is administered to the host
prior to the first administration of Plasmodium immunogen in the
vaccination regimen, usually at least 2 days prior to first dose,
and administration of the antimalarial continues, usually for at
least 30 days subsequent to the last dose in the regimen, such that
the level of antimalarial in the bloodstream of said host is
sufficient to prevent the signs, symptoms and pathology of malaria.
For example, as disclosed in Roestenberg (Id.), chloroquine may be
administered orally in two 300 mg doses starting 2 days prior to
the first exposure to SPZ and continuing in weekly 300 mg doses
until 1 month after the last exposure. It is a transformative
approach to human vaccination because it harnesses the infectious
agent's inherent replicative properties to amplify production of
protective immunogens spanning multiple developmental stages, and
then eliminates the infectious agent with an anti-infective drug
before the onset of disease. For either vaccine, an adjuvant that
increases the potency of the SPZ sporozoite would be of enormous
value.
[0033] Most of the technical hurdles in the development of malaria
vaccines comprising pharmaceutical compositions of live attenuated
sporozoites and live infectious sporozoites have now been
overcome--among them, aseptic production of sufficient quantities
of sporozoites isolated from attendant material using cGMP protocol
(See particularly U.S. Pat. No. 7,229,627; Hoffman, SL, et al.,
2010 Hum. Vac. 6:97-106--both explicitly incorporated herein by
reference).
[0034] Regarding a vaccine suitable for routine use in human
subjects that comprises live attenuated sporozoites, and live
infectious sporozoites, the sporozoites must be substantially
purified from the source from which they were produced.
Pharmaceutical compositions comprising substantially purified
sporozoites and excipient as well as methods of purifying
sporozoites are known in the art (U.S. Patent Publ. No.
2010/0183680. This publication is explicitly incorporated herein by
reference.
[0035] Plasmodium-species parasites are grown aseptically in
cultures as well as in vivo in Anopheles-species mosquito hosts,
most typically Anopheles stephensi hosts. Methods of axenically
culturing Plasmodium-species liver stage parasites (Kappe et al. US
Pub. 2005/0233435) and methods of producing attenuated and
non-attenuated Plasmodium-species sporozoites, particularly,
methods of growing and attenuating parasites in mosquitoes, and
harvesting attenuated and non-attenuated sporozoites are known in
the art and have been described (See: U.S. Pat. No. 7,229,627; U.S.
Pub. No. 2005/0220822).
[0036] PfSPZ Vaccine, a malaria vaccine comprising live attenuated
Plasmodium sporozoites without adjuvant, has been developed and is
in clinical trials (Hoffman S. L. et al., Development of a
metabolically active, non-replicating sporozoite vaccine to prevent
Plasmodium falciparum malaria. 2010 Hum. Vac. 6:97-106. DOI: 10396
[pii].). The vaccine has already been assessed in a first-in-humans
Phase I clinical trial in 80 healthy, malaria-naive adults at Naval
Medical Research Center and the University of Maryland (Epstein J.
E., et al. Supra.). The vaccine was administered intradermally (ID)
or subcutaneously (SC) to 80 volunteers with the primary goal of
establishing safety. Results of this dose-escalation study
demonstrated the PfSPZ Vaccine is safe, well tolerated, and without
breakthrough infections. Furthermore, the vaccine induced antibody
and T cell immune responses, and protected several volunteers.
However, as expected based on pre-clinical data, optimal immune
responses and protection were not achieved in this first trial and
studies in mice, rabbits, and monkeys have pointed the way toward
the next steps in the R&D process (Epstein, J. E. et al, Id.).
The Pf sporozoites in the PfSPZ Vaccine are highly potent. Based on
animal studies, it appears that the effectiveness of
pre-erythrocytic stage malaria vaccines correlates with the
induction, of interferon gamma producing CD8+ T cells in the liver.
It is expected that this immune response will eliminate the
Pf-infected liver cells. When rhesus monkeys were immunized
intravenously (IV) with the PfSPZ Vaccine, 4 months after the last
dose, 3% of all CD8+ T cells in the liver were specific for PfSPZ.
No such responses were seen in monkeys immunized by the SC route,
as was done in the first clinical trial. In mice, IV administration
of irradiated, purified, cryopreserved P. yoelii SPZ induced high
levels of protection at low doses; 88% (21/24) of mice were fully
protected after 3 doses of 2,000 irradiated PySPZ. When
administered SC or ID, as in the first clinical trial, immune
responses were 50 to 150 fold lower and it required 7 to 10 times
as many sporozoites to achieve high level protection.
[0037] It is anticipated that IV administration of the vaccine will
reduce the number of sporozoites required to provide protection
against subsequent challenge with pathogenic parasites and
effectively reduce the incidence of malaria among a group of
individuals exposed to pathogenic Plasmodium parasites. However,
even in mice, it currently requires 3 doses administered IV to
achieve high level protection, and it would be preferable to
achieve such protection with fewer doses. Furthermore,
administration by a parenteral non-IV route would be preferable.
Therefore, it is desirable to reduce the number of sporozoites per
dose, and perhaps the number of doses, required to confer
protective immunity by a parenteral non-IV route of administration.
These requirements provide the framework for identifying an
effective adjuvant that is compatible for use with live attenuated
sporozoites.
[0038] Prior to the discovery disclosed herein, no adjuvant had
been described as compatible with live attenuated Plasmodium
sporozoites. The successful development of a sporozoite-compatible
adjuvant to maximize the protective efficacy of parenterally
administered sporozoites is universally applicable to sporozoites
attenuated by all means, including radiation, chemicals or genetic
alteration, thereby enabling a highly effective vaccine for the
prevention of malaria delivered by a parenteral route (TV or
non-TV).
Methods of Making Glycolipids
[0039] The methods of synthesis of the glycolipid analogues of the
present invention are provided in Tsuji et al (U.S. Patent Publ.
No. 2007/02388871, explicitly incorporated by reference). Unlike
laboratory reagents for use in small animal studies, all biologic
substances must be manufactured under Good Manufacturing Practice
(GMP) conditions (Code of Federal Regulations Title 21, Part 211,
April 2009) before use in humans One of the practical advantages of
glycolipids is their straightforward synthesis pathway from
chemical compounds that are readily available at relatively low
cost. In order for glycolipids to function as effective adjuvants
that provide dose-sparing of vaccines against infectious diseases
with high prevalence in the developing world, low cost and ease of
manufacturing are desirable (Padte, et al., at page 4).
[0040] Five analogues of .alpha.-GalCer were chosen based on
enhanced stimulatory activity against human iNKT cells in vitro,
binding affinity to human and murine CD1d molecules, and binding
affinity to the invariant t cell receptor of human iNKT cells (Li,
X., et al., Design of a potent CD1d-binding NKT cell ligand as a
vaccine adjuvant 2010 Proc Natl Acad Sci USA 107: 13010-13015,
explicitly incorporated herein by reference).
[0041] The genus of the 5 analogues is shown schematically in
formula (1):
##STR00001##
where R is: (CH.sub.2).sub.5Ph(p-OMe); (CH.sub.2).sub.7Ph(p-OMe);
(CH.sub.2).sub.7Ph(p-F); (CH.sub.2).sub.10Ph(p-F); or
(CH.sub.2).sub.10Ph(p-CF.sub.3).
[0042] Each of these 5 analogues are designated as follows:
##STR00002##
formula (2) (designated as C18 in Li et al., and designated formula
101 in U.S. Pat. No. 7,923,013)
##STR00003##
formula (3) (designated as C22 in Li et al., and designated as
formula 102 in U.S. Pat. No. 7,923,013)
##STR00004##
formula (4) (designated as C23 in Li et al., and designated as
formula 104 in U.S. Pat. No. 7,923,013)
##STR00005##
formula (5) (designated as 7DW8-5 in Li et al., and designated as
formula 105 in U.S. Pat. No. 7,923,013)
##STR00006##
formula (6) (designated as 7DW8-6 in Li et al., and designated as
formula 108 in U.S. Pat. No. 7,923,013)
Administration
[0043] In certain embodiments, a pharmaceutical composition
comprising one or more species of live Plasmodium sporozoite-stage
parasites is co-administered with a glycolipid adjuvant. In some
embodiments, the co-administration is by the same or a different
route of administration. For example, a pharmaceutical composition
comprising one or more species of live Plasmodium sporozoite-stage
parasites administered by an intravenous, intramuscular,
intradermal, or subcutaneous route can be co-administered with a
glycolipid adjuvant administered by an intravenous, intramuscular,
intradermal, or subcutaneous route. In a further embodiment, an
antimalarial drug can be further co-administered by an intravenous,
intramuscular, intradermal, or subcutaneous route.
[0044] In some embodiments, the co-administration is concurrent,
e.g., as an admixture. In some embodiments, the co-administration
is sequential. In certain embodiments, the time between sequential
administration events is not greater than about one hour, not
greater than about 30 minutes, not greater than about 15 minutes,
not greater than about 10 minutes, or not greater than about 5
minutes between the administrations. In certain embodiments, the
time between co-administration events is 0-60 minutes between
administrations, 0-30 minutes between administrations, 0-15 minutes
administrations, 0-10 minutes between administrations, or 0-5
minutes between administrations.
[0045] In certain embodiments, a pharmaceutical composition
comprising one or more species of live Plasmodium sporozoite-stage
parasites is co-administered with one or more glycolipid adjuvants
and additionally in the presence of an antimalarial drug at
sufficient concentration in the bloodstream of the host to prevent
the signs, symptoms or pathology of malaria upon subsequent
exposure to pathogenic parasites. In other embodiments, a
pharmaceutical composition comprising one or more species of live
Plasmodium sporozoite-stage parasites and a glycolipid adjuvant are
administered on the same time course or administered on an
overlapping time course relative to an antimalarial drug, i.e., the
antimalarial drug is not co-administered. As described, in some
embodiments, the antimalarial drug was previously administered such
that the concentration of said drug in the bloodstream of said host
is sufficient to prevent the clinical manifestations of
malaria.
Pharmaceutical Compositions
[0046] In compiling the results of the experiments disclosed in
Examples 1 through 3, a combined 13% of BALB/c mice inoculated with
radiation attenuated P. yoelii sporozoites (irr PySPZ) were
protected from subsequent challenge by immunization with one dose
administered IV. This level of protection improved to 75% when
7DW8-5 (formula (5)) was first delivered to the subject mice by
intraperitoneal (IP) injection followed by the suboptimal IV dose
of radiation attenuated Py sporozoites. These data show that
7DW8-5, administered distally and IP, can reduce the number of
doses required to confer protection.
TABLE-US-00001 TABLE 1 Summary of experiments described in Examples
1-3 protected/ percent Regimen challenged protected irr PySPZ
delivered IV 4/30 13% irr PySPZ delivered IV + 30/40 75% 7DW8-5
delivered IP 7DW8-5 delivered IP 0/15 0% Infectivity controls 0/23
0%
[0047] While P. yoelii is a species of Plasmodium with a mouse host
range, it is widely used and generally considered a predictive
model of the behavior of human host range Plasmodium by those
skilled in the art (See e.g. Khan, Z. M. and J. P. Vanderberg
(1991) Role of Host Cellular Response in Differential
Susceptibility of Nonimmunized BALB/c Mice to Plasmodium berghei
and Plasmodium yoelii Sporozoites. Infect. and Immun.
59(8):2529-2534). Plasmodium species with human host range include
P. falciparum, P. vivax, P. ovale, P. knowlesi, and P.
malariae.
[0048] All of the references cited above, as well as all references
cited therein, are incorporated herein by reference in their
entireties.
EXAMPLES
Example 1
Immunization of BALB/c Mice with Fresh Unpurified Radiation
Attenuated Plasmodium yoelii Sporozoites
[0049] To determine if the adjuvant 7DW8-5 could decrease the
number of doses of irr PySPZ required to confer protection, a
suboptimal dosing regimen was performed. irr PySPZ (17NXL) were
dissected from the salivary glands of A. stephensi mosquitoes and
injected intravenously (N) into BALB/c mice as a single dose of
either of 5.times.10.sup.4 or 1.times.10.sup.4 sporozoites. Two
.mu.g of 7DW8-5 was concurrently administered intraperitoneally
(IP) in some mice. Fourteen days later, mice were challenged with
100 non-irradiated pathogenic P. yoelii sporozoites. Protection was
assessed by Giemsa-stained blood smears and was defined as the
complete absence of parasitemia 7 and 14 days post infection.
[0050] In the absence of adjuvant, 0/5 mice and 1/5 mice that
received 5.times.10.sup.4 and 1.times.10.sup.4 respectively were
protected. When administered with adjuvant, 3/5 and 4/5 mice were
protected, respectively. Adjuvant alone provided no protection,
indicating an antigen specific response.
TABLE-US-00002 TABLE 2 Example #1. Immunization with fresh,
unpurified radiation attenuated Py sporozoites # of BALB/c fresh,
unpurified irr 2 .mu.g #protected/ mice PySPZ IV 7DW8-5 IP
#Challenged 5 5 .times. 10.sup.4 - 0/5 5 5 .times. 10.sup.4 + 3/5 5
1 .times. 10.sup.4 - 1/5 5 1 .times. 10.sup.4 + 4/5 5 -- + 0/5 5
Infectivity controls - 0/5
Example 2
Immunization of BALB/c Mice with Fresh Unpurified Radiation
Attenuated Py Sporozoites
[0051] A second experiment was performed to confirm the results
shown in Example 1. Using the same method as Example 1, a single
dose of 1.times.10.sup.4 irr PySPZ did not protect any of 10 mice,
but when combined with adjuvant protected 7 of 10 mice. Again,
adjuvant in the absence of antigen had no effect.
TABLE-US-00003 TABLE 3 Example #2. Immunization with fresh,
unpurified radiation attenuated Py sporozoites # of fresh,
unpurified irr 2 .mu.g #protected/ BALB/c mice PySPZ IV 7DW8-5 IP
#challenged 10 1 .times. 10.sup.4 - 0/10 10 1 .times. 10.sup.4 +
7/10 5 -- + 0/5 10 Infectivity control - 0/10
Example 3
Immunization of BALB/c Mice with Fresh Purified Radiation
Attenuated Py Sporozoites
[0052] Using the single dose methodology of Example 1 with
radiation attenuated Py sporozoites that have been purified using
the purification procedure disclosed in Sim et al. (U.S. Pat. No.
8,043,625, incorporated in its entirety herein by reference) a
third experiment was performed. Using 1.times.10.sup.4 purified
sporozoites administered IV in a single dose, 3 out of 10 mice were
protected, whereas 7 of 10 mice were protected when the adjuvant
was concurrently administered T.
TABLE-US-00004 TABLE 4 Example #3. Immunization with purified
radiation attenuated Py sporozoites number of BALB/c fresh irr
PySPZ 2 .mu.g # protected/ mice IV 7DW8-5 IP # challenged 10 1
.times. 10.sup.4 + 9/0 unpurified 10 1 .times. 10.sup.4 - 3/10
purified 10 1 .times. 10.sup.4 + 7/10 purified 5 -- + 0/5 8
Infectivity controls - 0/8
Example 4
Rechallenge--Longevity of Protection
[0053] Fifteen weeks after their first challenge with fresh,
infectious P. yoelii sporozoites, the mice that had received a
single dose of 10.sup.4 irradiated P. yoelii sporozoites with or
without concurrent administration of adjuvant IP and were protected
in Experiment #1 were re-challenged by the intravenous inoculation
of 100 fresh, pathogenic P. yoelii sporozoites. Surprisingly, all
four mice that had been protected after a single inoculation of
10.sup.4 irradiated P. yoelii sporozoites with concurrent distal IP
administration of adjuvant were protected upon re-challenge. The
single mouse that had been protected after administration of
10.sup.4 irradiated P. yoelii sporozoites without adjuvant was not
protected upon re-challenge. All five infectivity controls
developed parasitemia (see Table 5). The protection after a single
inoculation of 10.sup.4 irradiated P. yoelii sporozoites with 2
.mu.g 7DW8-5 adjuvant was solidly sustained for at least 15
weeks.
TABLE-US-00005 TABLE 5 Example #4 -. Rechallenge Experiment.
Immunization with fresh, unpurified radiation attenuated Py
sporozoites Re-challenge 101 days (15 weeks) after #of fresh, First
Challenge 1.sup.st challenge BALB/c unpurified 2 .mu.g # protected/
# protected/ mice irr PySPZ IV 7DW8-5 IP # re-challenged #
re-challenged 5 1 .times. 10.sup.4 - 1/5 0/1 5 1 .times. 10.sup.4 +
4 4/4 5 -- + 0/5 5 Infectivity - 0/5 controls 5 Infectivity - 0/5
controls
Example 5
Single Dose Optimization
[0054] To determine the optimal single dose of attenuated
sporozoites (in conjunction with 7DW8-5 concurrently administered
distally IP) that confers protection by the IV route, mice were
injected IV with 0, 2500, 5000, 10,000 and 20,000 sporozoites. Two
.mu.g of 7DW8-5 were injected IP. As shown in Table 6, 80%
protection was achieved in the group of mice receiving 10,000
sporozoites.
TABLE-US-00006 TABLE 6 Example #5 - Dose Optimization Experiment.
Immunization with fresh, unpurified radiation attenuated Py
sporozoites number of fresh, unpurified irr 2 .mu.g 7DW8-5 #
protected/ BALB/c mice PySPZ IV IP # challenged 5 0 + 0/5 5 0.25
.times. 10.sup.4 + 0/5 5 0.5 .times. 10.sup.4 + 2/5 5 1 .times.
10.sup.4 + 4/5 5 2 .times. 10.sup.4 + 1/5 5 Infectivity controls -
0/5
Example 6
Two Dose Optimization--Intradermal
[0055] To determine whether protective immunogenicity of attenuated
sporozoites is maintained or enhanced in the presence of 7DW8-5,
mice were immunized by intradermal (ID) injection alone, with 2
doses at 2-week intervals of 15,000, 10,000, 5,000 and 2,500
sporozoites and unlike Examples 1-5, 1 .mu.g 7DW8-5 was mixed as a
composition with sporozoites and the composition of adjuvant and
sporozoites Was delivered ID at the base of the tail. With 2 doses
of 15,000 sporozoites in the presence of adjuvant 100% of the mice
were protected, and at lower doses of sporozoites 60-80% of the
mice were protected. In the absence of adjuvant, only 40% of mice
receiving 2 doses of 15,000 sporozoites were protected. This
demonstrates that 7DW8-5 is compatible with live attenuated
Plasmodium sporozoites, and the composition enhances the protection
provided by attenuated sporozoites alone.
TABLE-US-00007 TABLE 7 Example # 6 - Two dose Intradermal
Immunization. Immunization with fresh, unpurified radiation
attenuated Py sporozoites ID co-administered with adjuvant at base
of tail. fresh, 1 .mu.g # doses at 2- number of unpurified 7DW8-5
per week # protected/ BALB/c mice irr PySPZ ID mouse intervals #
challenged 5 1.5 .times. 10.sup.4 - two 2/5 5 1.5 .times. 10.sup.4
+ two 5/5 5 1.0 .times. 10.sup.4 + two 3/5 5 0.5 .times. 10.sup.4 +
two 4/5 5 0.25 .times. 10.sup.4 + two 3/5 5 0 + 0/5 8 Infectivity
0/8 controls
Example 7
Single Dose Immunization Intradermal
[0056] Protection conferred by a single dose of a composition of 1
.mu.g 7DW8-5 adjuvant and varying amounts of sporozoites delivered
ID was measured. For this 15,000 and 30,000 sporozoites were
administered to mice ID as described above. These doses were
partially protective at 60% and 40% respectively.
TABLE-US-00008 TABLE 8 Example #7 - Single dose Intradermal
Immunization Experiment. Immunization with fresh, unpurified
radiation attenuated Py sporozoites ID co-administered with
adjuvant at base of tail number of fresh, BALB/c unpurified 1
.mu.g7DW8-5 # doses at # protected/ mice irr PySPZ ID per mouse
2-week intervals # challenged 5 1.5 .times. 10.sup.4 + one 3/5 5 3
.times. 10.sup.4 + one 2/5 5 Infectivity - two 0/5 controls
Example 8
Single Dose Purified Sporozoites ID
[0057] The next step in development was to assess purified irr
PySPZ BALB/c/mice were immunized with in PySPZ purified as
described, in the presence or absence of 7DW8-5 adjuvant. Three out
of five (60%) mice were protected after immunization with 30,000
purified irr PySPZ mixed with 1 .mu.g adjuvant administered ID at
the base of the tail, and none were protected in the absence of
adjuvant. No protection was observed in mice receiving adjuvant
alone.
[0058] In the foregoing, the present invention has been described
with reference to suitable embodiments, but these embodiments are
only for purposes of understanding the invention and various
alterations or modifications are possible.
* * * * *