WO2002011757A2 - Compositions and methods for inhibiting transmission of malaria - Google Patents

Abstract

Compositions and methods that elevate serum levels of antibodies capable of selectively binding to at least one antigenic determinant of the mosquito midgut. When the antibody-containing serum is ingested by a feeding mosquito, the antibody provides an effective barrier to malarial ookinete penetration of, and encystment within the midgut wall. Further transmission of the parasite to a fresh host is significantly reduced or eliminated. Compositions containing one or more polyclonal or monoclonal antibodies, capable of selectively binding to an antigenic determinant of the mosquito midgut are also described. Alternative compositions contain antigenic determinants from the mosquito midgut. The compositions may alternatively contain nucleic acids that encode antigenic determinants of the mosquito midgut. When compositions containing antibodies, antigenic determinants from the mosquito midgut, or nucleic acids encoding the antigens are administered to animals or humans, antibodies specific for the mosquito midgut are induced and the serum level of the antibodies is raised. Mosquitoes feeding on the human or animal ingest the antibodies, which bind to the midgut wall, thereby inhibiting malarial transmission.

Description

COMPOSITIONS AND METHODS FOR INHIBITING TRANSMISSION OF MALARIA

This invention was made by the Centers for Disease Control, an agency of the United States Government. Therefore the United States Government has certain rights in this invention.

TECHNICAL FIELD The present disclosure relates in general to antigen or antibody compositions, and methods of using the compositions to block transmission of infectious disease, particularly malaria, through mosquitoes.

BACKGROUND OF THE DISCLOSURE Malaria is the most common human parasitical disease, infecting 300-500 million persons worldwide. It results in as many as 3 million deaths each year, mainly of African children. Although endemic throughout tropical and subtropical climatic regions, the increasing likelihood of global warming suggests that many more areas, previously free of the disease could see future outbreaks.

Besides the obvious cost in human suffering, malaria places major burdens on the already limited medical services of developing nations and severely impacts economic productivity. Tourism to countries where malaria is common is also harmed, reducing an important source of foreign exchange.

Indigenous populations can develop a degree of immunity to malaria, if they survive childhood bouts with the disease. Travelers from temperate zones to the infected regions, on the other hand, are more likely to fall victim unless they take active prophylactic measures well in advance of their visits. The most effective anti-malarial treatment is quinine derivative drugs administered before, during and after any possible exposure to parasite-bearing mosquitoes. Physical barriers to mosquito bites, such as sleeping nets and clothing, also reduce the chances of infection. Malaria has so far, however, successfully evaded any durable or prolonged vaccine protection.

Malaria is caused in humans by four species of the protozoal Plasmodium (P.) parasite: P. falciparum, P. vivctx, P. malariae and P. ovale and variants thereof. In mice, a common malarial parasite is P. berghei. There is widespread distribution and extensive geographic overlap of these species in the tropical and sub-tropical regions around the globe, although P. ovale is almost exclusively confined to the African sub-continent.

The essential insect vector for transmission of the parasite to humans is any of approximately .

20 species of mosquito, from a total of about 500 known species. The most prevalent malaria vectors designated by region are as follows: North America - Anopheles (An.) freeborni and An. qiiadrimaculatus; Central and South America - An. albimanus, An. albitarsus, An. aquasalis, An. bellator, An. cr zi, An. darlingi, An. freeborni, An. oswaldoi, An. pseudopunctipennis, An. punctimacula, and An. quadrimaculatus; Africa - An. arabiensis, An. funestus, An. gambiae, An. labranchiae, An. melas, An. multicolor, An. nili, and An. sergenti; Asia - An. culicifacies, An. farauti, An. fluviatilis, An. jeyporiensis, An. maculapennis, An. maculatus, An. minimus, An. sacharσvi, An. sinensis, An. stephensi, An. sundaicus, and An. superpictus. The most important species with regard to malaria transmission to humans are An. gambiae, An. culicifacies, An. funestus, An. stephensi, An. darlingi, and An. albimanus. The mosquito, however, is not merely a passive vector. It plays a crucial role in the complex multi-step and multi-form life cycle of the Plasmodium protozoa.

The cycle begins when an infected female mosquito bites an animal or human and injects saliva and the sporozoite form of the parasite into the blood stream. The sporozoites are rapidly transported to the liver where they attach to specific receptors. They then enter hepatic parenchymal cells and begin to asexually reproduce. The liver cells ultimately burst, and the merozoite stage of the parasite is released into the blood stream. This also signals the onset of the symptomatic phase of the disease, typified by fever and delirium. With P. vivax and P. ovale, some infected cells do not break open, and the parasite can remain dormant for considerable periods. When these cells finally do rupture and release their contents, there is a relapse in the disease that can occur many years after the initial infection. Circulating merozoites rapidly attach to receptors on the surface of erythrocytes, and enter the blood cells. They then develop into trophozoites that grow to occupy the entire cellular volume.

Plasmodium-specific immunodominant proteins also become incorporated into the erythrocyte cell membrane. Multiple nuclear divisions result in the formation of between 6 and 30 daughter merozoites, which are then released to infect other erythrocytes. Each cycle of merozoite replication lasts approximately 48 hours. In addition, some of the merozoites develop into gametocytes that do not attach to blood cells, and remain free in the blood stream.

A feeding female mosquito ingests gametocytes in a blood meal from an infected animal or human. The male and female gametocytes fuse once in the gut of the mosquito to form a zygote.

This matures to an ookinete that attaches to epithelial cells lining the gut, and then burrows through, and encysts within, the gut wall. Multi-nucleate division of the oocysts, of which typically there might be about 1 to 3 per insect, results in a mass of sporozoites. These enter the insect hemolymph, and migrate to the salivary glands. They remain there until injected into the next mammalian host when the insect feeds again.

P. falciparum is the most virulent form of malaria in humans and, if left untreated, can attain a mortality rate as high as 20%. Key to this high virulence rate are the parasite-induced changes to the surface of infected erythrocytes that allow them to bind to endothelial cells of small blood vessels of the brain, heart and other vital organs.

Efforts to control malaria have concentrated on limiting the population of mosquitoes, or on prophylactic measures that limit the progress of an infection. While insecticides, elimination of standing water necessary for the insect to breed, and measures to avoid being bitten have been successful, they have not been able to eradicate malaria entirely from an infected area.

Chemical prophylaxis is the principal weapon against infection, especially for travelers from temperate areas who have no naturally acquired immunity. The same drugs are also used therapeutically in the event that an infection is established. Mefloquine is the preferred drug for use in the tropics, and is reasonably tolerated and safe for pregnant females. It is effective against multidrag-resistant P. falciparum. Chloroquine is the drug of choice against drug-sensitive P. falciparum. While still generally effective as prophylactic agents, however, many strains of Plasmodium are now displaying resistance to the most commonly used chemotherapeutic agents. In an infected human, if the disease is not ultimately fatal, the onset of a specific immune response eventually limits the extent of symptoms and brings the disease under some degree of control. A victim that survives the initial onslaught of the parasite acquires cellular and humoral immunity, but notably only to the specific strain of the parasite, and then only to the parasitemia phase of the infection. Natural immune systems do not totally eradicate malaria from the body since the parasite can remain sequestered in hepatocytes and is protected from the host's immune system, ready to reemerge years later. Repeated outbreaks are not uncommon.

Conscripting the human immune system to neutralize malarial infections has proven problematic. There are multiple forms of the parasite in the life cycle, and each carries unique immunogenic determinants that can rapidly adopt mutational variations. The lack of major immunohistocompatability antigens on the surface of the erythrocytes, the potential of the parasite to express immunodominant antigens on the surface of the blood cells, and the antigenic diversity of the Plasmodium compromise the ability of the cellular immune system to directly attack the protozoa. It has proven difficult, therefore, to create an effective vaccine aimed solely to antigenic determinants carried by the parasite, that is effective against several, if not all, strains of the malarial parasite, and that affords long-term protection. For the forgoing reasons, therefore, there is an urgent need for an effective means to block the infective malarial life cycle that does not rely on directly attacking the parasite.

What is needed, therefore, is a composition that can be ingested by a mosquito to intervene in the development of the parasite and its interactions within the insect vector, thereby reducing the transmissibility of malaria from the insect host to a new mammalian host.

SUMMARY OF THE DISCLOSURE The present disclosure provides compositions and methods to reduce the transmissibility of malaria by a mosquito vector that solve problems described above. Disclosed compositions contain anti-mosquito midgut antibodies or immunogenic proteins or peptide antigens that elevate serum levels of antibodies specifically directed against one or more antigenic determinants of the mosquito midgut region. When antibody-containing blood or blood serum is ingested by a feeding mosquito, the antibody binds to the insect midgut and provides an effective barrier to malarial ookinete penetration of, and encystment within, the wall of the midgut, thereby inhibiting development of the malarial parasite and reducing transmission of the disease.

Blockage of ookinete penetration into the wall of the midgut inhibits development of the ookinete into sporozoites. The anti-mosquito midgut antibodies, therefore, interrupt the life cycle of the malarial parasite within the arthropod vector, thereby inhibiting sporozoite passage to the mosquito's salivary gland. This dramatically reduces or eliminates transmission of the parasite by the mosquito when biting the next animal or human.

A preferred embodiment of the composition described herein contains one or more polyclonal or monoclonal antibodies capable of binding to at least one antigenic determinant specific to the mosquito midgut. The antibodies are administered in a carrier readily consumed by a mosquito, such as blood or an aqueous solution.

In a second preferred embodiment, the composition is a mosquito midgut lysate containing at least one mosquito midgut-specific immunogenic determinant or epitope that, when administered with a pharmaceutically acceptable carrier to a human or an animal, will induce an immune response and elevate serum levels of antibodies specific for antigens of the mosquito midgut. Alternatively, the antigen is a partially purified or purified preparation of at least one mosquito midgut-specific determinant or epitope.

In yet another embodiment, the composition contains synthetic peptides whose amino acid sequences are similar or identical to those of the mosquito midgut antigens, combined with a pharmaceutically acceptable carrier for administration to a human or animal.

In a fourth preferred embodiment, the composition contains nucleic acid molecules having sequences that encode at least one mosquito midgut-specific protein or peptide. Administration of these nucleic acid sequences to a human or animal in an appropriate vector results in translation into mosquito midgut-specific peptides and proteins that then induce an immune response and produce serum levels of antibodies specific for the mosquito midgut region.

While not wanting to be bound by the following statement, it is believed that when the mosquito ingests the antibodies, the binding of antibody to a luminal surface antigen in the mosquito midgut specifically prevents, inhibits, or reduces attachment of the ookinete stage of the malarial parasite to the mosquito midgut wall. This blocks the development of the parasite to the oocyst and eventually the sporozoite form that would otherwise infect a fresh mammalian host and establish a new infectious cycle. It has been discovered that some antigenic determinants on the luminal surface of the mosquito midgut are common to many species of mosquito that act as vectors of the human malarial parasite. The antibodies of the compositions described herein, therefore, are especially effective against the development of the virulent human malarial parasite P. falciparum within mosquito species that harbor the Plasmodium parasite and provide an effective inhibitor to the transmission of animal and human virulent malarial parasites.

Accordingly, embodiments of the present disclosure provide safe and effective compositions and methods useful for reducing the transmission of malaria from mosquito to host by interfering with the development of the parasite within the mosquito.

An additional advantage is that the antibodies demonstrate an insecticidal effect. In certain embodiments, once attached to the insect midgut, they also disturb the normal functioning of the alimentary canal and reduce the viability of the insect resulting in increased mortality and reduced egg-laying capacity.

Other embodiments provide compositions and methods for reducing malarial disease using compositions that avoid the development of drug resistance. Yet another embodiment provides a method to increase serum levels of an antibody in a human or an animal to disrupt the ability of a mosquito feeding on the treated human or animal to transmit viable and infectious forms of the malarial parasite to another animal or human host.

Also provided are methods for interrupting the Plasmodium life cycle in the mosquito host. Other embodiments provide methods for treating large populations of mature mosquitoes to render them incapable of transmitting viable Plasmodium.

Embodiments also provide methods and compositions having mosquito insecticidal effects through disrupting mosquito physiology.

Other features and advantages will become apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 is a pair of graphs illustrating the effect on survival of feeding anti-midgut antibodies to An. gambiae mosquitoes.

DETAILED DESCRIPTION OF THE DISCLOSURE

The compositions and methods described herein contain or generate antibodies, preferably circulating antibodies within a human or animal, that specifically bind to antigens of the insect midgut region when ingested by a feeding mosquito. The antibodies confer transmission blocking immunity ^• by interrupting the malarial parasite life cycle in the insect host. Additionally, they reduce vector abundance through an insecticidal effect.

In a preferred embodiment, the composition includes an antiserum or an antibody specific for at least one antigenic determinant of the mosquito midgut. Both partially purified and pure antibody compositions may be employed. The composition may further include a pharmaceutical carrier. A preferred antigen is administered as a DNA vaccine. Alternatively, the composition contains peptides or proteins capable of inducing the formation of antibodies that, when ingested by a feeding mosquito, specifically bind to antigens of the insect midgut. Preferably, the^" composition-includes at least one protein or peptide with an amino acid sequence identical or similar to the sequence of at least one antigenic determinant of the mosquito midgut. The composition may further include a pharmaceutical carrier or carrier peptide or protein. The composition described herein can include a nucleic acid in a vector, or not in a vector that, when administered to an animal or human, is expressed as a protein or peptide with an amino acid sequence identical or similar to the sequence of at least one antigenic determinant of the mosquito midgut. The composition may further include a pharmaceutical carrier. Once inoculated into an animal, expression of the nucleotide sequence generates peptides and an immune response to the peptides, thereby raising antibodies against the mosquito midgut. The methods described herein include methods for producing anti-mosquito midgut antibodies in a human or animal and methods for inhibiting malaria transmission. In a preferred antibody production method, a composition containing at least one antibody capable of binding to an antigenic determinant of the midgut of the mosquito is administered to a human or animal. In an alternative antibody production method, a composition containing an immunogen that produces antibodies to mosquito midgut is administered to a human or animal. The antibodies are ingested by a feeding mosquito and bind to the midgut of the insect. Although it is intended that the feeding mosquito ingest the antibodies with a blood meal, this does not exclude feeding the insects on a nutrient solution containing the midgut-specific antibodies.

The administered or induced circulating antibodies ingested by a feeding mosquito block the development of the malarial parasite in the mosquito. Once in the midgut of the insect, the antibodies bind to the midgut region of the alimentary canal. Attachment of the ookinete form of the malarial parasite is thereby prevented and the life cycle of the parasite interrupted. Transmission of the malarial parasite to a new human is therefore disrupted. The method for inhibiting malaria transmission therefore involves feeding mosquitoes a food source containing anti-midgut antibodies.

Definitions The terms "immunogenic compound" or "immunogenic composition" as used herein refer to any compound or composition that includes at least one species of antibody specific for one antigenic determinant or a peptide, protein or antigen capable of inducing the formation of such an antibody in a recipient animal or human. The immunogenic compound or composition is a polyclonal antibody, or at least one monoclonal antibody. The immunogenic compound or composition also includes a nucleic acid molecule that, when expressed in an animal or human, encodes a peptide or protein that induces the formation of antibodies. The immunogenic compound or composition may also include a pharmaceutically acceptable component that will enhance the ability of the composition to produce effective serum levels of an antibody.

"Peptides," "polypeptides" and "oligopeptides" are chains of amino acids (typically L-amino acids) in which carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the carbon of one amino acid and the amino group of the carbon of another amino acid.

"Immunogen" refers to an entity or fragment thereof that can induce an immune response in a mammal. The term includes immunogens and regions responsible for antigenicity or antigenic determinants.

"Antigen" refers to an entity or fragment thereof capable of specifically binding to the antigen-binding site of an antibody. The term includes immunogens and regions responsible for antigenicity or antigenic determinants. The term "antigenic determinant" refers to a region of an antigen molecule that is specifically recognized by an antibody, and is bound by said antibody.

The term "epitope" refers to a minimal region of an antigenic determinant capable of being recognized by an antibody. The phrases "specifically binds to a peptide" or "specifically immunoreactive with", when referring to an antibody, refers to a binding reaction that is determinative of the presence of a peptide, or an antibody to a peptide, in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular peptide and do not bind in a significant amount to other proteins present in the sample. Specific binding to a peptide under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

The term "nucleic acid or "oligonucleotide" is used herein to mean deoxyribonucleic acid (DNA including cDNA) or ribonucleic acid (RNA) in either single- or double-stranded form. Unless otherwise limited, "nucleic acid" encompasses known analogs of natural nucleotides that can function in a manner similar to the naturally occurring nucleotides. The phrase "nucleic acid sequence encoding" refers to a nucleic acid sequence that directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA sequence that is transcribed into RNA and the RNA sequence that is translated into the protein. The nucleic acid sequence can include both the full-length nucleic acid sequence as well as non-full length sequences derived from the full-length sequence. It will be understood by those of skill that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

Antigenic Compositions

Antigenic compositions described herein include antigens, antigenic determinants or epitopes of the mosquito midgut. The antigens included are in the form of:

(a) Tissue Lysates: Midguts from sugar-fed female mosquitoes are dissected in a buffer and homogenized.

(b) Partially Purified Antigens: Antigens specific to the mosquito midgut may be partially purified preparations of total protein lysates. The partially purified proteins may be isolated by protein separation techniques such as, but not limited to, centrifugation, gel chromatography, electrophoresis, salt precipitation, pH-dependant precipitation, reverse phase liquid chromatography, affinity chromatography or by other methods or combinations thereof known to one of ordinary skill in the art. (c) Purified Polypeptides or Peptides: Antigens of the compositions described here may be purified preparations of proteins, or of peptides derived therefrom. The proteins are purified from the mosquito midgut by methods well known to one of ordinary skill in the art. Peptides may be derived from a purified polypeptide by restrictive proteolytic digestion, mechanical cleavage such as by sonication, chemical synthesis of ohgopeptides or by other methods known to one of ordinary skill in the art. A peptide with antigenic activity specific to the mosquito midgut may be purified by gel electrophoresis, reverse phase liquid chromatography, gel chromatography, or by any means that will separate peptides on the basis of such parameters as, but not limited to size, pi, amino acid sequence, electric charge. By way of example, antigens of the mosquito midgut that can be used to induce the immunogenic response disclosed herein have apparent molecular weights of between 150 kDa and 7 kDa as visualized by SDS-PAGE; antigenic fragments of large proteins, and/or carbohydrates or protein/carbohydrate complexes, also may serve as immunogens.

Typically, the immunogenic peptide of interest is at least about 3 amino acids. More typically the peptide is 5 amino acids in length. Preferably, the fragment is 10 amino acids in length, and more preferably the fragment is 15 amino acids in length or greater. Often, the fragment is about 20 amino acids in length. Immunogenic conjugates are typically prepared by coupling the peptide to a carrier protein as a fusion protein or, alternatively, they are recombinantly expressed in an immunization vector. Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.

(d) Nucleic Acid: Antigenic compositions as described herein may be nucleic acid molecules encoding a polypeptide or peptide specific to the mosquito midgut. The nucleic acid, when administered to an animal or human, is expressed to yield the corresponding antigenic polypeptide or peptide. The antigenic nucleic acid molecule is DNA or RNA and may be purified or linked to other nucleic acid molecules that are necessary for the expression of the encoded polypeptide or peptides, such as but not limited to, promoters, enhancers or other regulatory regions. After administering the nucleic acid to an animal or human, the nucleic acid may be randomly incorporated into the nucleus of at least some cells of the animal or human to whom the nucleic acid antigenic composition is administered. The nucleic acid can be incorporated into the genomic DNA of the recipient cell or remain in the cytoplasm, wherein it is translated to a peptide sequence.

Immunogenic Conjugates

Immunogenic conjugates containing one or more of the antigenic polypeptides or peptides described above, covalently attached to a carrier protein, are also provided. Suitable carrier proteins include, but are not limited to, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(D-lysine: D-glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine, etc. When the peptide and carrier protein are relatively short in length (i.e., less than about 50 amino acids), they are preferably synthesized using standard chemical peptide synthesis techniques. When both molecules are relatively short, a chimeric molecule is optionally synthesized as a single contiguous polypeptide. Alternatively, the peptide and the carrier molecule can be synthesized separately and then fused chemically.

Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the immunogenic conjugates provided herein. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc, 85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nded. Pierce Chem. Co., Rockford, 111. (1984).

Alternatively, the immunogenic conjugates are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide- carrier protein immunogenic conjugate, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein. Techniques sufficient to guide one of skill through such procedures are found in, e.g., Berger and Kimmel, Sambrook, Ausubel, at the citations provided above. While the peptide and carrier molecule are often joined directly together, one of skill will appreciate that the molecules may be separated by a spacer molecule (e.g., a peptide) consisting of one or more amino acids. Generally, the spacer will have no specific biological activity other than to join the immunogenic peptide to the carrier protein, or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.

Once expressed, recombinant immunogenic conjugates can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)). Substantially pure compositions of about 50 to 95% homogeneity are preferred, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents.

One of skill in the art will recognize that after chemical synthesis, biological expression, or purification, the immunogenic conjugates may possess a conformation substantially different than the native conformations of the constituent peptides. In this case, it is often necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art. Antibody Compositions

Antibody compositions described herein are capable of binding at least one antigenic determinant of the mosquito midgut. The antibody or antibodies, when bound to the midgut-specific antigen block the passage of the malarial parasite ookinete into the wall of the mosquito midgut where it would normally encyst.

Antibody compositions include polyclonal antisera or partially purified or purified immunoglobulins therefrom, capable of binding to at least one midgut specific antigen. Alternatively, the antibody composition contains at least one monoclonal antibody capable of recognizing at least one mosquito midgut specific antigen. The antibody may be selected from immunoglobulin classes such as IgA, IgG or IgM, and isotopes thereof, such as but not limited to, IgGl, IgG2a and IgG3. The monoclonal antibodies may be raised against antigens on the luminal surface of the midgut region or against intracellular antigenic determinants of the mosquito midgut. A variety of monoclonal antibodies raised therefrom can detect proteinaceous antigens of approximately 10 kDa to at least 200 kDa. In a preferred embodiment, the monoclonal antibody detects antigen on the luminal surface of at least some epithelial cells lining the midgut lumen. The most preferred antibody is the monoclonal antibody designated MG25E, which is produced by a hybridoma deposited with the American Type Culture Collection (ATCC) on , as ATCC Accession No. .

Antibody Production Methods

Antibodies are raised to the proteins or peptides described above, including individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring forms and in recombinant forms. Additionally, antibodies are raised to the proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies are also generated. Many methods of making antibodies are known to those skilled in the art. The following discussion is presented as a general overview of the techniques available; however, one of skill in the art will recognize that many variations upon the following methods are known.

Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogenic, preferably a purified peptide, a peptide coupled to an appropriate carrier (e.g., GST, keyhole limpet hemocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Patent No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogenic preparation is monitored by taking test bleeds and determining the titer of reactivity to the peptide of interest. When appropriately high titers of antibody to the immunogenic are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the peptide is performed where desired.

Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified peptides, or screened for agonistic or antagonistic activity. Specific monoclonal and polyclonal antibodies will usually bind with a Krj of at least about 0.1 mM, more usually at least about 50 mM, and most preferably at least about 1 mM or better. Often, specific monoclonal antibodies bind with a KD of 0.1 mM or better.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, and the like. Descriptions of techniques for preparing such monoclonal antibodies are found in, e.g., Stites et al. (Eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256: 495-497. Summarized briefly, this method proceeds by injecting an animal with an immunogen. The animal is sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secretes a single antibody species directed to the immunogen. In this manner, the individual antibody species are products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance..

Alternative methods of immortalization include transformation with Epstein-Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and the yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The antigens and antibodies in the compositions described herein are used with or without modification, and include chimeric antibodies such as humanized murine antibodies. Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341 : 544-546. Frequently, the antigens and antibodies will be labeled by joining, either covalently or non covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuchdes, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include the following: U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;

4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029- 10033.

The antibodies provided herein can be used in affinity chromatography for isolating the antigenic determinants including, but not limited to, the mosquito midgut recognized by the antibody. Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified peptides are released. In addition, the antibodies can be used to screen expression libraries for particular expression products including, but not limited to, for example, mosquito midgut proteins. Usually, the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding.

Methods of Administering Antigen or Antibody to Human or Animal to Inhibit Transmission of the Malaria Parasite

The antigen compositions described herein are administered to a human or animal to provide an immune response. An "immune response" as used herein is one that produces a sufficient antibody titer that it reduces the ability of a mosquito, having ingested a blood meal derived from the recipient of the composition, to transmit malarial parasites to a new mammalian host. An amount sufficient to accomplish this is defined as an "immunogenically effective dose." Amounts effective for this use will depend on the composition, the manner of administration, the weight and general state of health of the subject, and the judgment of the prescribing physician. For peptide compositions, the general range for the initial immunization (that is for therapeutic or prophylactic administration) is from about 500 μg to about 1 gm of peptide for a 70 kg patient, followed by boosting dosages of from about 100 μg to about 1 gm of the peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition. For nucleic acids, typically 30-1000 μg of nucleic acid is injected into a 70 kg patient, more typically about 150-300 μg of nucleic acid is injected into a 70 kg patient followed by boosting doses as appropriate.

The immunogenic or pharmaceutical compositions described above may contain a pharmaceutically acceptable carrier for administration to a human or animal. Such compositions are suitable for use in a variety of drug delivery systems. Suitable formulations are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, and 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference.

The antigen or antibody compositions can be administered together in different combinations. The compositions are suitable for single administrations or a series of administrations. When given as a series, inoculations subsequent to the initial administration are given to boost the immune response and are typically referred to as booster inoculations.

The pharmaceutical compositions provided herein are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, compositions are provided for parenteral administration that include a solution of the agents described above dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%.

Nucleic acid encoding an immunogenic protein may be introduced into humans or animals to obtain an immune response to the immunogenic peptides that the nucleic acid encodes. See, Wolff, et al., Science 247: 1465-1468 (1990), the teachings of which are incorporated herein by reference. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods. Such manufacturers include the SIGMA Chemical Company (St. Louis, MO), R&D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, CA, and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill. The nucleic acid compositions, whether RNA or DNA, are isolated from biological sources or synthesized in vitro. The nucleic acids in some embodiments are present in transformed or transfected whole cells, in transformed or transfected cell lysates, or in a partially purified or substantially pure form.

In vitro amplification techniques suitable for amplifying sequences for generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990)

Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.

Small nucleic acids (less than 100 nucleotides in length) are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20): 1859- 1862, e.g., using an automated synthesizer, as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Nucleic acids can also be custom made and ordered from a variety of commercial sources known to persons of skill. Purification, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom. 255:137-149.

The sequence of synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560.

The immunogenic compositions and methods provided herein prevent the transmittal of at least some virulent strains of the malarial parasite to humans and animals. The compositions described herein are not rendered ineffective by variation in the antigenic determinants of the parasite itself. The immunogenic compositions and the methods of administering thereof focus instead on binding to midgut antigenic determinants that are common to at least some species of the mosquito. These determinants are required by the ookinete of the malarial parasite for passage into the wall of the mosquito midgut. Once in the midgut wall the ookinete develops into the infectious sporozoite form. An additional advantageous consequence is that the antibodies, once attached to the insect midgut, may also disturb the normal functioning of the alimentary canal and reduce the viability of the insect.

It has been unexpectedly discovered that polyclonal antisera raised against antigens of the midgut region of the mosquito blocks passage across the insect midgut wall by malarial parasite species virulent to humans, including P. falciparum and P. vivax. The mosquito species include, but are not limited to, An. stephensi, An. freeborni, An. albimanus, An. gambiae and An. far auti. Surprisingly, a monoclonal antibody specific for a single protein expressed on the luminal surface of the anopheline vector A. gambiae, effectively blocked transfer of P. falciparum and P. vivax across the wall of the midgut in other mosquito species. Therefore, polyclonal and the monoclonal antibodies immunoreactive with the midgut of one species of mosquito unexpectedly are of sufficient stability, durability and cross-reactivity when ingested by a mosquito of the same or another species to effectively block the progress of malarial ookinetes to the encystment stage of their development. The preferred antibody is a polyclonal antibody preparation specific for the midgut region of the anopheline mosquito. A further preferred antibody is a monoclonal antibody having the capacity to block transmission of various malarial parasite species by several species of the mosquito. More preferably, the antibody is a monoclonal antibody specific for an antigen present on or in the mosquito midgut. Most preferably, the antibody is a monoclonal antibody immunoreactive with an antigen of the midgut of the A. gambiae mosquito, and recognizes an epitope on a proteinaceous antigen of about 105 kDa that is found in the midgut of a plurality of mosquito species.

Monoclonal antibodies (mAbs) are preferably generated using donor B-cells from mice immunized with midgut lysates from a mosquito species selected from, but not limited to, Anopheles gambiae, A. stephensi, A. freeborni A. albimanus and A. farauti. Mosquito midgut lysates are prepared by dissecting and homogenizing mosquito midguts. Mice are immunized with the lysate in an adjuvant such as Freund's Complete Adjuvant, and preferably boosted at least once. Spleen cells from hyperimmune mice are fused with myeloma cells in the presence of polyethylene glycol 1500, and subsequent culture supernatants are screened for the presence of anti-midgut antibodies by ELISA using techniques well known to those skilled in the art.

The protein, peptide or epitope recognized by the monoclonal antibody is one that is necessary for the ookinete stage of at least some plasmodial species to attach and pass through the wall of a mosquito midgut.

In the most preferred embodiment, the monoclonal antibody is the antibody designated MG25E, produced by the hybridoma deposited with the ATCC under Accession No. , and prevents the development of malarial parasites, including, but not limited to P. falciparum, P. vivax, P. malariae, and P. ovale.

It will be understood by those ordinarily skilled in the art that the polyclonal or monoclonal preparations described herein may consist of intact immunoglobulins, or fragments thereof that retain the ability to bind to a specific epitope. For example, the immunoglobulin fragment may be the Fv region of an immunoglobulin, or a single-chain antibody wherein the antigen-binding regions of an immunoglobulin heavy and a light chain are contiguous in a single polypeptide. Typically, the administered antibody preparation will be retained in the circulating blood stream of the recipient human or mammal long enough for an effective dose to be ingested by a feeding mosquito. The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are intended neither to limit nor define the invention in any manner.

Example 1: Blockage of Transmission oft. falciparum in Mosquitoes Fed Immune Serum To test the potential of midgut-based transmission blocking immunity against the human parasite, P. falciparum, immune sera with known blocking activity against P. berghei were pooled and fed to A. stephensi mosquitoes in conjunction with infective P. falciparum gametocyte cultures. Mosquitoes were examined at the appropriate times thereafter for ookinetes, oocysts, and sporozoites. Pooled immune sera was mixed with normal human sera in a 1 : 1 ratio, and administered with infectious P. falciparum gametocyte cultures to A. stephensi mosquitoes via membrane feeders. Gametocyte cultures containing either normal mouse or normal human sera served as negative controls. Unfed mosquitoes were removed and engorged mosquitoes were incubated at 24°C. Six to eight mosquitoes in each experimental group were dissected about 30 hours post-infection and blood meals were examined for ookinetes. Eight to eleven days later, approximately 50 mosquitoes from each experimental group were dissected and their midguts were examined for oocysts. On days 18-21 post-feeding, remaining mosquitoes were dissected and salivary glands were squashed and examined for sporozoites. Appropriate transformations (logι₀X + 1) were performed for statistical comparison of means (t-tests and analyses of variance) and computation of confidence intervals. Infection rates were compared by chi-square analysis.

There were no differences between the ookinete prevalences or densities in mosquitoes fed either anti-midgut immune sera or normal mouse sera, showing that the anti-berghei immune sera did not specifically inhibit P. falciparum gametogenesis, fertilization, or zygote transformation. Both immune and non-immune mouse sera had a slight, albeit statistically insignificant (χ2 and analysis of variance ANOVA) inhibitory effect on these early sporogenic events, typical of when non-human sera are used in P. falciparum gametocyte cultures. Parasite prevalences remained stable between ookinete and oocyst lifestages for both normal mouse (about 60%) and normal human (about 80%) sera control groups of mosquitoes. Parasite densities in both control groups decreased approximately 10-fold during the ookinete-oocyst transition. Oocyst prevalence and density in the anti-midgut immune fed group were significantly less than in either control group (χ2's > 12, df = 1, p-valύes < 0.005) (t-values > 5, df = 98, p-values < 0.005). Similarly, the sporozoite rate in immune serum-fed mosquitoes was significantly less than in either control group (χ2's > 10, df = 1, p-values < 0.005, the sporozoite rate as used here is the number of mosquitoes with sporozoites per number of dissected mosquitoes). Transmission in immune-fed mosquitoes was effectively blocked.

Example 2: Generation of Monoclonal Antibodies Against Mosquito Midgut Lysates

Monoclonal antibodies (mAbs) were generated using donor B-cells from mice immunized with Anopheles gambiae midgut lysates. A. gambiae mosquito midguts were dissected and snap frozen in sterile phosphate buffered saline (PBS) containing the protease inhibitors iodoacetamide (5.0 mM, Sigma Chemical Co. St. Louis, MO), pepstatin A (1.0 μM, Sigma), leupeptin (1.0 μM, Sigma), EDTA (0.5 μM, Gibco BRL, Rockville, MD), PEFABLOC SC™ (1,0 M, Roche Molecular Biochemicals, Indianapolis, IN; PEFABLOC SC™ is a trademark of Pentaphann AG, Basel Switzerland) and aprotinin (1%, Sigma). These proteins were thawed and immediately emulsified with Freund's Complete Adjuvant (FCA). Balb/c mice were immunized with 100 μg of the midgut proteins in Freund's Complete Adjuvant, and boosted twice with 50 μg of midgut proteins in Freund's Incomplete Adjuvant. Spleen cells from hyperimmune mice were fused with Sp2/0 myeloma cells in the presence of polyethylene glycol 1500, by techniques known to those skilled in the art. After two weeks, culture supernatants were screened for the presence of anti-midgut antibodies by ELISA.

A total of 17 mAbs with the isotypes IgGl, IgG2a, and IgG3 reacted with A. gambiae midgut lysates. Western blotting and confocal microscopy were used to select those mAbs most likely to block transmission.

Example 3: Selection of Monoclonal Antibodies for Transmission Blocking Tests

A. gambiae mosquito midguts were dissected and frozen in sterile phosphate buffered saline (PBS) containing protease inhibitors. After heating in sample buffer at 65°C for 15 minutes, approximately 10 g of midgut protein (10 midguts) were electrophoretically separated on a 5-20 % SDS-polyacrylamide gradient gel under reducing conditions. Proteins were transferred to a nitrocellulose membrane and membrane strips were incubated overnight at 4°C with 5 ml of mAb supernatant. Membrane strips were washed with PBS containing 0.3% Tween 20 (PBS-TW), and incubated 1 hour at about 20°C with peroxidase-conjugated goat anti-mouse IgG antibodies, diluted 1 :3000 in PBS-TW. After washing with PBS-TW, bound antibodies were detected using 3,3' diaminobenzidine and 30% hydrogen peroxide. Relative molecular weights were estimated using broad range molecular weight markers. The Western blot analyses of the various monoclonal antibodies (mAbs) against A. gambiae midgut lysates yielded various electrophoretic patterns (Table 1).

Table 1. ANTI-ANOPHELES GAMBIAE MIDGUT MONOCLONAL ANTIBODIES

One group, including monoclonal MG25E, recognized a single band at about 105 kDa. (Banding Pattern A, Table 1). Other monoclonal antibodies recognized one band at approximately 10 kDa and one band at 105 kDa (Banding Pattern B), a single band at 10 kDa (Banding Pattern D) or ^ multiple bands (Banding Pattern C). Based on these observations, monoclonal antibody MG25E was selected for subsequent transmission blocking bioassays.

Example 4: Histological Localization of The Antigens Specifically Detected by The Anti-Mosquito Midgut Monoclonal Antibodies

Mosquito midguts were examined as cryosections and everted whole mounts. For sections, midguts were imbedded in TISSUE-TEK™ O.C.T. compound, frozen in an isopentane liquid nitrogen bath and cut using a cryostat. (TISSUE-TEK™ O.C.T. is a formulation of water soluble glycols and resins used as an embedding compound for cryosections at low temperatures; TISSUE- TEK™ is a trademark of Sakura Finetek U.S.A., Inc., Torrance, CA.) Whole mount staining was performed by placing individual midguts into a small drop of PBS on glass slides. Midguts were slit longitudinally and opened, to expose the luminal surface. Sections and whole mounts were fixed in absolute methanol and treated with anti-mosquito midgut antigen mAbs by incubating serial 2-fold dilutions of anti-mosquito midgut mAb MG25E with midgut cryosections for 30 minutes at 37°C in a humidity chamber, rinsed with phosphate buffered saline (PBS) and stained with a fiuorochrome- conjugated goat anti-mouse antibody to visualize mAbs bound to mosquito midgut antigens. Three- dimensional orientation of the antibody staining was visualized by taking optical sections through the tissue in steps of 0.1-0.2 μm with a Zeiss (LSM 2) confocal laser scanning microscope. The microscope was configured with 25 mW Ar and He-Ne lasers with 488, 514, and 543 maximum lines and software for image acquisition of x-y, and z series scan three-dimensional visualization.

Confocal microscopic analyses of cryosectioned and everted whole A. stephensi midguts revealed different immunostaining patterns. Many of the mAbs identified what appeared to be internal cytoskeleton elements. Monoclonal antibody MG25E displayed immunofluorescent staining of cryosectioned midguts consistent with the surface localization of antigen. Whole-mounts of everted midguts stained with MG25E exhibited surface immunostaining of some, but not all, of the luminal midgut epithelial cells. The pattern of "patchy" immunofluorescence on luminal membrane borders was consistent with the distribution of Ross cells although no separate verification of cell type was performed. Identical patterns were observed with A. gambiae midguts stained with MG25E.

Example 5: Transmission Blockage of P. falciparum Gametocytes Using Monoclonal Antibody MG25E Transmission blocking effects of MG25E were assayed by feeding mosquitoes on infectious

P. falciparum gametocytes via membrane feeders containing mAb MG25E. Normal human sera and a biologically irrelevant mAb, NYLS3, directed against P. yoelii liver stage antigen, served as separate negative controls. Approximately 1.5 ml of each mixture was added to individual water jacketed membrane feeders and placed on the screened tops of separate cages, each containing approximately 300 female Anopheles stephensi mosquitoes. After 1 hour, feeders were removed and unfed mosquitoes destroyed. Mosquitoes were provided with sugar and a moistened pledge and incubated thereafter at 25 °C. Mosquitoes were periodically examined for ookinete, oocyst and sporozoite content.

The mAb MG25E had no effect on ookinete production compared to controls (p- values>0.05). However, oocyst production was significantly affected. There was greater than a 50% reduction in oocyst prevalence (χ2 = 34.6, df = 2, p < 0.0001) and a 95% reduction in oocyst density (ANOVA, f = 53.8, df = 2,184, p < 0.0001) in mosquitoes that ingested MG25E compared to control groups. Similarly, the sporozoite infection rate of the MG25E group was essentially half that in the control groups (%2 = 10.0, df = 2, p < 0.010). Thus, mAb MG25E interfered with passage of P. falciparum ookinetes through the mosquito midgut.

Example 6: Transmission Blockage oft. vivax Gametocytes Using Monoclonal Antibody MG25E To determine if mAb MG25E would block transmission of the second most important human malaria parasite, a transmission blocking immunity bioassay using P. vivax was conducted.. In addition, three other species of anopheline mosquito were tested to determine if MG25E, originally generated against A. gambiae-deήved antigens and biologically active in A. stephensi, would also block transmission in other more distantly related Anopheles species. Transmission blocking effects of MG25E were assayed by mixing 1.0 mg mAb per ml of chimpanzee blood containing P. vivax gametocytes. Normal chimpanzee sera and another mAb, IBl 1 (IgGl) directed against P. vivax blood stage parasites, served as separate negative controls. Approximately 1.0 ml of each mixture was added to individual water jacketed membrane feeders and fed to about 60 females each of A. stephensi, A. freeborni, A. albimanus, and A. farauti. At the appropriate time thereafter, mosquitoes were examined for ookinete and oocyst content.

There was no significant treatment effect (p-values > 0.05) on ookinete production within any of the four mosquito species, as shown in Table 2.

Table 2. Transmission blocking effect of the anti-mosquito midgut monoclonal antibody

MG25E on Plasmodium vivax development in four species of co-infected Anopheles mosquitoes.

--Ookinete-- —Oocyst— -Sporozoite--

Treatment P D P D P

—Anopheles stephensi—

Normal Chimpanzee Sera 100% a 132 a 100% a 138 a 100% a

(5) (58-297) (11) (102-184) (24)

Control mAb IBl 1 100% a 138 a 100%a 30b 100% a

(6) (58-318) (11) (19-50) (22)

Anti-mosquito mAb MG25E 100% a 293 a 79% a 3 c 69% b

(5) (185-463) (19) (1-5) (13)

—Anopheles freeborni—

Normal Chimpanzee Sera 80% a 40 a 91% a 18 a ND

(5) (3-472) (11) (3-88)

Control mAb IB 11 67% a 28 a 100% 13 a ND

(3) (-1-6,797) (12) (7-24)

Anti-mosquito mAb MG25E 100% 123 a 17% (b) 0.1b ND

(2) (51-293) (18) (0.01-0.3)

—Anopheles albimanus— •

Normal Chimpanzee Sera 83% a 63 a 89% a 55 a ND

(6) (7-535) (9) (13-223)

Control mAb IBl 1 80% a 41 a 91% a 9 b ND

(5) (2-505) (11) (3-24)

Anti-mosquito mAb MG25E 80% 46 a 33% b l c ND

(5) (2-626) (9) (-0.1-3.6)

—Anopheles farauti—

Normal Chimpanzee Sera 100% a 178 a 86% a 35 a ND

(3) (104-305) (28) (17-72)

Control mAb IB 11 67% 16 a 83% a 3b ND

(3) (1-1,678) (24) (2-5)

Anti-mosquito mAb MG25E 100% a 159 a 45% b 0.7b ND

(3) (76-332) (11) (0.1-1.7)

D = Density P = Prevalence

Infection prevalences were expressed as percentages. The number of mosquitoes examined is in parentheses. Parasite densities were expressed as geometric means (95% confidence limits). Letters denote the results of statistical comparisons for each mosquito species. Within each lifestage (vertical columns), prevalences, and densities among the three treatments that have the same letter do not differ at the 0.05 level of significance. ND represents not determined. MAb IBl 1 was a mouse monoclonal antibody against P. vivax blood-stage parasites.

With normal chimpanzee sera, the conversion efficiencies of P. vivax ookinetes to oocysts (based on tage-specific densities) were essentially 100% in the natural vectors, A. stephensi and_^ ^" albimanus.

Control mAb LB11, generated against P. vivax blood stage antigens did not affect oocyst prevalence but it significantly reduced oocyst densities in all mosquito species, except A. freeborni (analysis of variance ANOVA, p-values < 0.05). This indicates that P. vivax ookinetes share a common epitope with blood-stage of the parasites. This epitope may have limited potential as a TBI target antigen. Monoclonal antibody MG25E, on the other hand, had significantly greater TBI activity than did control antibody IB 11. (Table 3.)

Table 3. Transmission blocking effect of mouse anti-mosquito midgut immune sera on Plasmodium falciparum development in Anopheles stephensi mosquitoes.

—Ookinete-- - Oocyst— -Sporozoite—

Treatment P D P D P

A

Normal Human Sera 83% a 45 a 90% a 4.27 a 53% a

(6) (5-369) (50) (3.09-5.79) (15)

Naϊve Mouse Sera 62% a 17 a 59% b 1.16 b 44% a

(8) (1-139) (51) (0.72-1.70) (32)

Anti-mosquito midgut 62% a 27 a 2% c 0.01 c 4% b immune sera (8) (4-169) (50) (-0.01-0.04) (23)

B

Normal Human Sera 83% a 92 a 82% a 12 a 56% a ⁱ.

(6) (6-1,346) (55) (8-18) (36)

Control mAb NYLS-3, Y 100% a 567 a 77% a 12 a 61% a

(10) (320-1,005) (66) (8-19) (49)

Anti-mosquito mAb MG25E 100% a 184 a 32% b 0.5 b 31% b

(10) (178-188) (76) (0.3-0.8) (51)

D = Density

P = Prevalence

MAb NYLS-3, Y was a mouse monoclonal antibody against P. yoelii liver stage parasites. Infection prevalences were expressed as percentages. The number of mosquitoes examined is in parentheses. Parasite densities were expressed as geometric means (95% confidence limits). Letters denote the results of statistical comparisons. Within each lifestage (vertical column), prevalences, and densities among the three treatments that have the same letter do not differ from one another at the 0.05 level of significance.

In A. stephensi, there was a 20% reduction in oocyst prevalence in the MG25E group compared with the normal chimpanzee sera group. There was also over a 50-fold reduction in oocyst density (t = 12.4, df = 28, p < 0.0001), showing that MG25E had a strong inhibitory effect on P. vivax ookinete passage through the midgut. When sample sizes were increased for salivary gland dissections, the sporozoite infection rate in the MG25E group was significantly less than the controls (χ2 = 7.5, df = 1, p < 0.01). The transmission blocking effect of MG25E was more extensive in the other three Anopheles species, reducing significantly both oocyst prevalence and density over those of the control groups (p-values < 0=05). Oocyst prevalences were-reduced 50 to 80% in A. freeborni, A.- albimanus and A. farauti. Thus, MG25E blocked transmission of P. vivax in multiple species of mosquitoes. Example 7: Transmission Blockage oft. vivax Gametocytes Using Monoclonal Antibodies MG25E, MG24C andMG4B

The transmission-blocking activities of two anti-midgut mAbs were compared with MG25E against P. vivax. Monoclonal Antibody Production

Anopheles gambiae mosquito midguts were dissected and snap frozen in sterile phosphate buffered saline (PBS) plus protease inhibitors, iodoacetamide (5.0 mM), pepstatin A (1.0 μM), leupeptin (1.0 μM), EDTA (0.5 μM), PEFABLOC (1.0 M) and aprotinin (1%) as described in more detail in Example 2, above. Balb/c mice were immunized with 100 μg of midgut proteins in Freund's Complete Adjuvant and boosted twice with 50 μg of midgut proteins in Freund's Incomplete Adjuvant. Spleen cells from hyperimmune mice were fused with Sp2/0 myeloma cells in the presence of polyethylene glycol 1500, by known techniques. After two weeks, culture supernatants were screened for the presence of anti-midgut antibodies by ELISA. Positive cultures were expanded and subcloned by limiting dilution. Transmission-Blocking Assay

For P. falciparum assays, polyclonal sera were mixed 1 : 1 with naϊve sera (human, or mouse depending on experiment) and administered together with infectious P. falciparum gametocyte cultures to mosquitoes via membrane feeders. Monoclonal antibody, MG25E, was assayed by mixing 0.67 mg mAb per ml of human blood containing infectious P. falciparum gametocytes. Gametocyte cultures containing control serum (human or mouse), and separately, a biologically irrelevant mAb (NYLS3, directed against P. yoelii liver-stage antigen), served as negative controls. For the P. vivax assays, monoclonal antibodies MG25E, MG24C, or MG4B were assayed by mixing 1.0 mg mAb per ml of chimpanzee blood containing P. vivax gametocytes. Normal chimpanzee sera and an irrelevant antibody, either IBl 1 (IgGl), directed against P. vivax blood-stage parasites, or a commercial polyclonal IgG mouse antibody (Sigma) served as separate negative controls. For all experiments, unfed mosquitoes were removed and engorged mosquitoes were incubated at 24°C. For each experimental group, mosquitoes were dissected and bloodmeals examined for ookinetes (27-30 hours post-infection for P. falciparum and 24-28 hours for P. vivax). Eight to eleven days later, mosquitoes for each experimental group were dissected and midguts were examined for oocysts. On days 18-21, the remaining mosquitoes were dissected and salivary glands examined for sporozoites. Experimental Protocol

The three antibodies, MG25E, MG24C, and MG4B were tested in An. stephensi and An. gambiae mosquitoes. Normal chimpanzee serum and a commercial polyclonal IgG mouse antibody were used as controls. As in previous experiments with P. falciparum, no significant effect on ookinete production was found in the two mosquito species. Control mouse polyclonal IgG antibody did not affect oocyst prevalence, but it slightly reduced oocyst intensity. However, the transmission- blocking activity of midgut-specific antibodies was highly significant. There was a 20% reduction in oocyst prevalence of An. stephensi given MG25E with the bloodmeal compared with those given normal chimpanzee serum. However, this difference did not reach a level of statistical significance (p>0.2). The reduction in prevalence of oocysts in mosquitoes fed MG24C was 93% (pO.OOl), and for those fed MG4B, the reduction was 45% (p<0.05). Of the anti-midgut antibody-fed mosquitoes that were positive for oocysts, the intensity of oocyst infection was also significantly lower. The percent reduction in intensity of infection by antibodies MG25E, MG24C or MG4B was 83%, 99%, and 98.6%, respectively (pO.OOOl).

The effects of anti-midgut mAbs on oocyst prevalence and intensity were more pronounced in An. gambiae mosquitoes, against which the mAbs were originally raised. Oocyst prevalence was reduced by 66%, 100%, and 100% m ^' An. gambiae given MG25E (pO.OOl), MG24C (p .01), or MG4B (pO.OOl) with a bloodmeal. There was a significant decrease in the number of oocysts in the mosquitoes fed antibody MG25E (>98% reduction, pO.OOOl), with 100% oocyst reduction seen in mosquitoes fed MG24C or MG4B. The normal mouse polyclonal antibodies had a slight, albeit not statistically significant, inhibitory effect on the sporogonic events in An. gambiae. These results indicate that there are probably several epitopes on the mosquito midgut that can be utilized in the successful development of midgut-based transmission-blocking immunity vaccines. These data further suggest that carbohydrate moieties on the midgut lumen may be important in the ookinete/midgut interaction.

Example 8: Insecticidal Effects of Monoclonal Antibodies MG25E, MG24C andMG4B on An. stephensi

A potential advantage of mosquito antigen-mediated transmission-blocking immunity is that these transmission-blocking antibodies also interfere with normal physiologic processes in the mosquito, and can result in reduced survival time and reduction in egg-laying capacity.

The effect of anti-midgut antibodies on mosquito survival and fecundity in An. stephensi was assessed. The addition of MG25E, MG24C, or MG4B to the bloodmeal increased mortality to 17%, 24%, and 7%, as compared with 5% in the chimpanzee serum-fed mosquitoes. In a follow-up experiment designed to determined the effects of anti-midgut antibodies on fecundity, An. stephensi mosquitoes were allowed to feed on blood supplemented with normal chimpanzee serum of midgut antibodies, MG25E, MG24C or MG4B. Compared with the number of eggs per female in the mosquitoes fed normal chimpanzee sera, addition of the anti-midgut antibodies resulted in a significant reduction in the number of eggs. These results suggest that anti-mosquito antibodies that exhibited transmission-blocking activity also reduced mosquito fecundity. Reduced fecundity may lead to lower overall mosquito abundance and further augment the reduction in malaria transmission conferred by the transmission-blocking immunity and the mortality effects described above.

Example 9: Insecticidal Effects of Monoclonal Antibodies MG25E andMG24C on An. gambiae

Cohorts of An. gambiae mosquitoes were fed normal human blood containing either monoclonal antibody MG25E, MG24C, NYLS3 (an irrelevant monoclonal antibody), or normal human serum. Each cohort was monitored for daily for survival. The results of a representative experiment are shown in Figure 1A. The percent mortality for the cohort of the mosquitoes fed human blood containing monoclonal antibody MG25E was greater than 80% by day 3 post bloodmeal as compared to 30% in control cohorts. The experiment was repeated using only monoclonal antibody MG25E (with appropriate controls), with similar results; the results of this experiment are shown in Figure IB.

In both experiments, monoclonal antibody MG25E, and to a lesser extent MG24C, is effective at reducing the survival of mosquitoes.

A transmission-blocking immunity approach based on mosquito antigens has several advantages over an approach based on parasite antigens. First, the intervention can work against different species of malaria parasites transmitted by different species of mosquitoes. Second, mosquito midgut-based transmission-blocking immunity has the added benefit of potentially decreasing mosquito survivorship and/or fecundity. Third, anti-midgut antibodies may also disrupt mosquito digestion/absorption enough to retard normal oocyst development in previously infected mosquitoes. The indirect effects on mosquito survivorship, fecundity, and parasite development may be cumulative and could substantially impact malaria transmission.

The disclosures of all publications cited in this application are hereby incorporated by reference in their entireties in order to describe more fully the state of the art to which this disclosure pertains. Modifications and variations of the present compositions and methods will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.

Claims

CLAIMS We claim:

1. An antibody preparation specific for a mosquito midgut having a luminal surface and epithelial cells included thereon, and which when ingested by a mosquito reduces transmission of an infectious biological agent.

2. The antibody preparation of claim 1, that when ingested by a mosquito reduces prevalence and/or density of oocysts of a malarial parasite in the mosquito.

3. The antibody preparation of claim 1 , comprising a polyclonal antibody capable of binding to the mosquito midgut.

4. The antibody preparation of claim 1, comprising a monoclonal antibody capable of binding to a mosquito midgut.

5. The antibody preparation of claim 4, wherein the monoclonal antibody is produced by hybridoma MG25E, American Type Culture Collection (ATCC) Accession No: , and wherein the monoclonal antibody is specific for an antigenic determinant on the luminal surface of the mosquito midgut.

6. An immunogenic composition comprising a pharmaceutically acceptable carrier and an immunologically effective preparation of a mosquito midgut having a luminal surface and epithelial cells included thereon, and which is capable of generating antibodies in a human or an animal that are specific for an antigenic determinant of the mosquito midgut.

7. The immunogenic composition of claim 6, wherein the immunologically effective preparation is a partially purified antigenic determinant of the mosquito midgut.

8. The immunogenic composition of claim 6, wherein the immunologically effective preparation is a purified antigenic determinant derived from the mosquito midgut.

9. An immunogenic composition comprising a pharmaceutically acceptable carrier and a nucleic acid encoding an antigenic determinant of a mosquito midgut having a luminal surface and epithelial cells included thereon, and which, when expressed in an animal or a human, is capable of generating antibodies in the human or an animal that are specific for antigenic determinants of the mosquito midgut.

10. A method of reducing the ability of a mosquito to transmit an infectious biological agent to a human or an animal by:

(a) increasing the serum level of an antibody in an animal or human so that the antibody is capable of being ingested by a mosquito taking a blood meal; and

(b) inoculating a mosquito with the circulating antibody when the mosquito ingests a blood meal.

11. The method of claim 10, wherein the antibody is a polyclonal antibody.

12. The method of claim 10, wherein the antibody is a monoclonal antibody.

13. The method of claim 10, wherein the monoclonal antibody is produced by the hybridoma MG25E, American Type Culture Collection (ATCC) Accession No: .

14. A method of reducing the ability of a mosquito to transmit a malaria parasite to a human or an animal by:

(a) inoculating the animal with a pharmaceutically acceptable carrier and an immunologically effective composition that will increase the serum level of an antibody in a human or an animal specific for antigenic determinants of the midgut region of a mosquito; and

(b) inoculating a mosquito with the antibody when the mosquito ingests a blood meal.

15. The method of claim 14, wherein the immunologically effective composition is a preparation containing at least one antigenic determinant from the mosquito midgut.

16. The method of claim 14, wherein the immunologically effective phaπnaceutical composition is a nucleic acid encoding a protein antigenic determinant of the mosquito midgut, wherein the nucleic acid is expressed in the human or animal and the expressed protein antigenic determinant is released to the blood stream to generate an immune response.