Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-26
2004-11-09
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S243000, C435S320100, C435S069700, C435S070100, C435S071100, C424S191100, C424S268100, C424S272100, C536S023100, C536S023500, C536S023700
Reexamination Certificate
active
06815183
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a parasite protein, and more particularly to
Plasmodium
sp.
chitinase
and uses thereof.
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description and throughout the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Defining molecular targets for drug or vaccine intervention remains a key strategy for developing new ways to prevent and treat malaria, a disease that exacts an enormous social and economic toll worldwide. A number of investigators have proposed transmission-blocking vaccines as one component of an overall program of malaria control (Kaslow 1997). Such vaccines are designed to induce antibodies in humans that, when ingested by the mosquito along with a
Plasmodium
-containing blood meal, interfere with the development of the parasite within the mosquito midgut. Animal models of transmission-blocking vaccines, based primarily on two
P. falciparum
zygote/ookinete surface proteins, Pfs25 and Pfs28, have demonstrated proof of principle (Gozar et al. 1998), but results of human clinical trials have not been reported to date.
A
Plasmodium
ookinete-secreted enzyme,
chitinase
(E.C. 3.2.1.14), has been demonstrated to be another target of blocking malaria transmission from humans to mosquitoes (Shahabuddin et al. 1993).
Chitinases
are found in prokaryotes and eukaryotes (Flach et al. 1992); their biologic roles include cell wall modification (e.g. fungi (Kuranda and Robbins 1991), Entamoebae (Willagomez-Castro et al. 1992) and filaria parasites (Fuhrman and Piessens 1985)), carbon source degradation (e.g.
Streptomyces
spp. (Ni and Westpheling 1997; Robbins et al. 1988),
Serratia marcescens
(Roberts and Cabib 1982), and Vibrio spp. (Keyhani and Roseman 1996)), and plant and fungal host defense against chitin-containing pathogens (Flach et al. 1992). One other protozoan pathogen of man,
Leishmania donovani
, the agent of human visceral leishmaniasis, is known to use a
chitinase
in its life cycle (Schlein et al. 1991; Shakarian and Dwyer 1998). The
Leishmania chitinase
is thought to disrupt the sand fly cardiac valve, allowing amastigotes to be regurgitated from the midgut into the skin of the vertebrate host. The
Leishmania chitinase
is not thought to function in invasion of the arthropod vector per-se (Schlein et al. 1992). In contrast,
Plasmodium chitinase
is thought to be required for the parasite to invade the mosquito midgut after being taken up in a blood meal (Shahabuddin et al. 1993). Because of its critical biological function in the life cycle of the malaria parasite, the
Plasmodium chitinase
is a potential target for blocking transmission from the vertebrate host to the mosquito vector (Shahabuddin et al. 1993).
The potential importance of
chitinase
in malaria parasite biology was first suggested by a transmission electron micrograph showing the
P. gallinaceum
ookinete penetrating and appearing to focally degrade the chitinous peritrophic matrix (PM) in the
Aedes aegypti
midgut (Sieber et al. 1991). The PMs of the
Plasmodium
vectors
Anopheles gambiae
(which carries human malaria parasites) and
A. aegypti
(which carries avian malaria parasites) are composed of chitin, a &bgr;-1,4-linked polymer of GlcNAc, with intercalated proteins including trypsins and peritrophins (Perrone and Spielman 1988; Shen and Jacobs-Lorena 1997; Shen and Jacobs-Lorena 1998).
P. gallinaceum
ookinetes secrete active
chitinase
(Huber et al. 1991; Vinetz and Kaslow 1998). Although
chitinases
are found throughout the prokaryote and eukaryote kingdoms, the biological function of
Plasmodium chitinases
must be different, because ookinetes do not contain chitin and there is no evidence that ookinetes use chitin or mono- or oligomers of GlcNAc as a carbon source.
Chitinases
are critical for allowing the parasite to escape the mosquito midgut, as evidenced by the observation that addition of the
chitinase
inhibitor allosamidin to a blood meal prevents oocyst development (Shahabuddin et al. 1993). Both
P. gallinaceum
in
A. aegypti
and
P. falciparum
in
A. freeborni
fail to develop into oocysts in the presence of this inhibitor. This effect could be completely reversed by enzymatic degradation of the peritrophic matris (PM) in vivo, by adding exogenous
chitinase
to the blood meal. These observations demonstrated that a
chitinase
is necessary for malaria parasites to invade the mosquito and initiate sporogonic development.
Because of intrinsic biologic interest and the potential for
Plasmodium chitinases
to be targets of interfering with malaria transmission (for a review of malaria transmission-blocking vaccines and the potential of
Plasmodium chitinases
as targets, see: Kaslow 1993; Shahabuddin and Kaslow 1993), a need exists for the identification of the malarial parasite
chitinase
.
SUMMARY OF THE INVENTION
To this end, the subject invention provides an isolated nucleic acid molecule encoding a
Plasmodium
sp.
chitinase
. The invention also provides an oligonucleotide complementary to at least a portion of the mRNA encoding the
Plasmodium
sp.
chitinase
.
The isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the
Plasmodium
sp.
chitinase
results in production of
Plasmodium
sp.
chitinase
in a host cell. Expression of the oligonucleotide in a host cell results in decreased expression of the
Plasmodium
sp.
chitinase
.
The invention further provides methods of screening a substance for the ability of the substance to modify
Plasmodium
sp.
chitinase
function, and a method of obtaining DNA encoding a
Plasmodium
sp.
chitinase
.
Further provided is an isolated nucleic acid molecule encoding a
Plasmodium
sp.
chitinase
, wherein the nucleic acid molecule encodes a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence. The second amino acid sequence is as shown in SEQ ID NO:3 or SEQ ID NO:4.
The invention further provides a DNA oligomer capable of hybridizing to a nucleic acid molecule encoding a
Plasmodium
sp.
chitinase
. The DNA oligomer can be used in a method of detecting presence of a
Plasmodium
sp.
chitinase
in a sample, which method is also provided by the subject invention.
The invention also provides an isolated
Plasmodium
sp.
chitinase
, a composition thereof, and antibodies or antibody fragments specific for the
Plasmodium
sp.
chitinase
. The antibodies and antibody fragments can be used to detect the presence of the
Plasmodium
sp.
chitinase
in samples. The subject invention further provides a method of producing an antibody specific for a
Plasmodium
sp.
chitinase
in a host. The method comprises selecting the isolated
Plasmodium
sp.
chitinase
or an antigenic portion thereof and introducing the selected
Plasmodium
sp.
chitinase
or antigenic portion thereof into a host to induce production of an antibody specific for
Plasmodium
sp.
chitinase
in the host. Further provided is an isolated
Plasmodium
sp.
chitinase
encoded by a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence, the second amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:4.
The subject invention further provides a method of preventing infection of mosquitoes by
Plasmodium
sp., the method comprising exposing the
Plasmodium
sp. to an amount of a compound effective to interfere with function of
Plasmodium
sp.
chitinase
, thereby preventing infection of the mosquitoes by the
Plasmodium
sp.
Further provided is a method of preventing transmission of malaria by a mosquito feeding on a subject that may harbor
Plasmodium
sp. organisms. The method comprises administering to the subject an
Baskar Padma
Rogalskyj & Weyand LLP
Smith Lynette R. F.
The Board of Regents of the University of Texas System
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