Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1997-10-20
2001-11-13
Mosher, Mary E. (Department: 1643)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S268100, C424S272100, C530S350000, C930S210000
Reexamination Certificate
active
06316000
ABSTRACT:
BACKGROUND OF THE INVENTION
Malaria continues to exact a heavy toll on humans. Between 200 million to 400 million people are infected by
Plasmodium falciparum
, the deadliest of the malarial protozoans, each year. One to four million of these people die. Approximately 25 percent of all deaths of children in rural Africa between the ages of one and four years are caused by malaria.
The life cycle of the malaria parasite is complex. Infection in man begins when young malarial parasites or sporozoites are injected into the bloodstream of a human by a mosquito. After injection the parasite localizes in liver cells. Approximately one week after injection, the parasites or merozoites are released into the bloodstream to begin the erythrocytic phase. Each parasite enters a red blood cell in order to grow and develop. When the merozoite matures in the red blood cell, it is known as a trophozoite and, when fully developed, as a schizont. A schizont is the stage when nuclear division occurs to form individual merozoites which are released to invade other red cells. After several schizogonic cycles, some parasites, instead of becoming schizonts through asexual reproduction, develop into large uninucleate parasites. These parasites undergo sexual development.
Sexual development of the malaria parasites involves the female or macrogametocyte and the male parasite or microgametocyte. These gametocytes do not undergo any further development in man. Upon ingestion of the gametocytes into the mosquito, the complicated sexual cycle begins in the midgut of the mosquito. The red blood cells disintegrate in the midgut of the mosquito after 10 to 20 minutes. The microgametocyte continues to develop through exflagellation and releases 8 highly flagellated microgametes. Fertilization occurs with the fusion of the microgamete and a macrogamete. The fertilized parasite, which is known as a zygote, then develops into an ookinete. The ookinete penetrates the midgut wall of the mosquito and develops into an oocyst, within which many small sporozoites form. When the oocyst ruptures, the sporozoites migrate to the salivary gland of the mosquito via the hemolymph. Once in the saliva of the mosquito, the parasite can be injected into a host, repeating the life cycle.
Malaria vaccines are needed against different stages in the parasite's life cycle, including the sporozoite, asexual erythrocyte, and sexual stages. Each vaccine against a particular life cycle stage increases the opportunity to control malaria in the many diverse settings in which the disease occurs. For example, sporozoite vaccines fight infection immediately after injection of the parasite into the host by the mosquito. First generation vaccines of this type have been tested in humans. Asexual erythrocytic stage vaccines are useful in reducing the severity of the disease. Multiple candidate antigens for this stage have been cloned and tested in animals and in humans.
However, as drug-resistant parasite strains render chemoprophylaxis increasingly ineffective, a great need exists for a transmission-blocking vaccine. Such a vaccine would block the portion of the parasite's life cycle that takes place in the mosquito or other arthropod vector, thus preventing even the initial infection of humans. Several surface antigens serially appear on the parasite as it develops from gametocyte to gamete to zygote to ookinete within the arthropod midgut (Rener et al.,
J. Exp. Med
. 158: 976-981, 1983; Vermeulen et al.,
J. Exp. Med
. 162: 1460-1476, 1985). Although some of these antigens induce transmission-blocking antibodies, their use in developing transmission blocking vaccines may be limited. For instance, the antigens may fail to generate an immune response in a broad segment of the vaccinated population. Others may only produce partial blocking of transmission.
Thus there is a need to develop transmission-blocking vaccines which induce high, long lasting antibody titers and which can be produced in large amounts at low cost. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides biologically pure Pfs230 polypeptides which preferably have an epitope capable of eliciting a transmission blocking immune response. The sequence of the full length protein is set forth in SEQ. ID. No. 2. The invention also provides recombinantly produced Pfs230 and isolated nucleic acids which encodes the polypeptides. The sequence of a nucleic acid which encodes the full length protein is set forth in SEQ. ID. No. 1.
Also disclosed are expression vectors comprising a promoter operably linked to a nucleic acid which encodes Pfs230 as well as cells comprising the vectors. In one embodiment, the expression vector is capable of directing expression in
E. coli.
The invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and Pfs230 in an amount sufficient to induce a transmission blocking immune response in a susceptible organism, such as a human. The Pfs230 is preferably an immunologically active fragment of the full length protein. Methods of preventing transmission of malaria comprising administering to a susceptible organism the pharmaceutical compositions are also disclosed.
DEFINITIONS
The term “Pfs230” refers to proteins expressed on the surface of
Plasmodium falciparum
gametocytes which have a molecular weight of about 360 kDa before processing. The term encompasses native proteins as well as recombinantly produced proteins that induce a transmission blocking immune response. It also includes immunologically active fragments of these proteins. “Immunologically active fragments” are those portions of the full length protein which comprise epitopes capable of eliciting a transmission blocking immune response or which are recognized by transmission blocking antibodies.
A “susceptible organism” is a Plasmodium host that is susceptible to malaria, for example, humans and chickens. The particular susceptible organism or host will depend upon the Plasmodium species.
The phrases “biologically pure” or “isolated” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. Typically, a protein is substantially pure when at least about 95% of the protein in a sample has the same amino acid sequence. Usually, protein that has been isolated to a homogenous or dominant band on a polyacrylamide gel, trace contaminants in the range of 5-10% of native protein which co-purify with the desired protein. Biologically pure material does not contain such endogenous co-purified protein.
Two sequences (either nucleic acids or polypeptides) are said to be “substantially identical” if greater than about 85% of the sequences are shared when optimally aligned and compared. Greater identity of more than about 90% is preferred, and about 95% to absolute identity is most preferred.
Another indication that nucleic sequences are substantially identical is if they hybridize to the same complementary sequence under stringent conditions. Stringent conditions will depend upon various parameters (e.g. GC content) and will be different in different circumstances. Generally, stringent conditions for nucleic acids isolated from
Plasmodium falciparum
are those in which the salt concentration is at least about 0.2 molar and the temperature is at least about 55° C.
REFERENCES:
patent: 4707445 (1987-11-01), McCulchan et al.
Kumar et al, Molecular and Biochemical Parasitology 53: 113-120, 1992.*
Williamson et al (Biochemistry 296:359-362, 1992.*
Williamson et al, Molecular and Biochemical Parasitology 75: 33-42, 1995.*
Riley et al, Parasite Immunology 17: 11-19, 1995.*
Murphy et al, Parasitology 100: 177-183, 1990.*
Sandhu et al, Vaccine 12: 56-64, 1994.*
Williamson, K.C., et al., “Immunoaffinity Chromatography Using Electroelution,”Biochemistry,296:359-362, 1992.
Williamson, K. et al. (1992) “Cloning and Expression of Plasmodium falciparum transmission blocking target antigen Pfs230”, (Abstract of the 41st Annual Meeting
Kaslow David C.
Williamson Kim C.
Mosher Mary E.
The United States of America as represented by the Department of
Townsend and Townsend and Crew LLC
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