WO2010078466A2 - Sand fly salivary proteins with anti-complement activity and methods of their use - Google Patents

Abstract

It has been surprisingly discovered that a sand fly salivary gland polypeptide, or a nucleic acid sequence encoding the polypeptide, can function as an inhibitor of complement activation. The present disclosure provides for methods of treatment of disorders associated with an immune response associated with increased complement activation utilizing compositions comprising the sand fly salivary gland polypeptides of the disclosure. Also provided herein is the use of sand fly salivary gland anti-complement proteins to block transmission of parasites from sand flies to humans and animals.

Description

SAND FLY SALIVARY PROTEINS WITH ANTI-COMPLEMENT ACTIVITY AND METHODS OF THEIR USE

REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No. 61/142,098, filed December 31, 2008, which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

The disclosure relates to methods of using sand fly salivary proteins, or nucleic acid sequences encoding these proteins, to inhibit activation of the complement system in a subject. More specifically, this disclosure relates to sand fly salivary proteins that act as an inhibitor of components of the complement system. The disclosure also relates to the use of sand fly salivary gland anti-complement proteins to block transmission of parasites from sand flies to humans and animals.

BACKGROUND

The complement system is a biochemical cascade of events that supplements a subject's immune system and is therefore a very important first line of defense against pathogens. The complement system consists of a number of small proteins which normally circulate in the plasma in an inactive state. These proteins are cleaved by proteases when the system is activated. The cleaved proteins in turn activate other proteins. This pattern of sequential activation results in an expanding cascade of activity.

Activation of the complement system encompasses three different pathways - the classical pathway, the alternative pathway, and the lectin pathway. While the components for each pathway are distinct, each one results in the development of a membrane attack complex (MAC), an enzyme complex which is incorporated into bacterial cell walls and induces the lysis of pathogens. In addition, each of these pathways has as its by-products a number of anaphylatoxins - small peptides which contribute to an inflammatory response. Abnormal activation of the complement system has the potential to cause unnecessary inflammatory reactions and damage to cells in host tissues. To prevent excessive activation, the complement system is tightly regulated. The complement system is, however, involved in many pathologies and syndromes. For example, it is believed that the complement system might play a role in many diseases with an immune component, such as asthma, lupus erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, and ischemia-reperfusion injuries. The classical complement pathway in many cases is responsible for complement-mediated tissue damage. A selectively specific and effective inhibitor of the classical pathway would be desirable if it could avoid inhibition of the alternative pathway. Such an approach would maintain anti-microbial defenses while minimizing complement- mediated tissue damage. Such a treatment would be particularly useful in immune- compromised subjects. Thus, there is a need for agents that can be used to interact with one or more of the components of the complement cascade and regulate the complement system.

SUMMARY OF THE DISCLOSURE

It is surprisingly discovered that a polypeptide secreted from the salivary gland of the sand fly Lu. longipalpis has potent anti-complement activity. The sand fly anticoagulant polypeptide, referred to as LJM19, has an amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55 and is encoded by nucleic acid residues 82-360 of SEQ ID NO: 56. Thus, methods are provided herein for using the disclosed sand fly salivary gland anti-complement polypeptides, and polynucleotides encoding the anti-complement polypeptides, to inhibit the activity of a component of the complement system in vitro or in vivo.

The methods include inhibiting a component of the classical complement pathway in a sample in vitro, comprising contacting the sample with an effective amount of a polypeptide, wherein the polypeptide has at least 90% (for example, at least 95% or 98%) sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55, thereby inhibiting (for example, selectively inhibiting) the component of the classical complement pathway. Inhibiting a component of the classical complement pathway comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% inhibition, compared to a sample that has not been contacted with the polypeptide.

In another embodiment of the methods, a subject is treated for a disorder associated with increased complement activation by (a) selecting a subject with a disorder associated with increased complement activation, and (b) administering to the subject a therapeutically effective amount of a polypeptide having at least 90% (for example, at least 95% or 98%) sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55, thereby preventing or treating the disorder associated with increased complement activation in the subject. In some embodiments, treating the subject comprises inhibiting lysis of a cell sample obtained from the subject by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, compared to a subject who has not been administered a therapeutically effective amount of the polypeptide.

In other methods, a subject is treated for a disorder associated with increased complement activation by (a) selecting a subject with a disorder associated with increased complement activation, and (b) administering to the subject a therapeutically effective amount of a nucleic acid sequence encoding a polypeptide having at least 90% (for example, at least 95% or 98%) sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55, thereby preventing or treating the disorder associated with increased complement activation in the subject. In one embodiment, the nucleic acid sequence comprises the sequence set forth as residues 82-360 of SEQ ID NO: 56, or a degenerated variant thereof. The nucleic acid may be operably linked to an expression control sequence, wherein the expression control sequence is a promoter. The promoter can be an inducible or a constitutive promoter.

Also provided herein are methods for preventing the development of a Leishmania parasite in a sand fly, by administering to a subject an immunologically effective amount of a polypeptide having at least 90% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55, wherein antibodies produced in the subject specifically bind the polypeptide set forth as residues 23-115 of SEQ ID NO: 55, and wherein the antibodies, when ingested by the sand fly, prevent Leishmania development in the sand fly, thereby preventing the development of the Leishmania parasite in the sand fly. In particular embodiments, the subject is a dog or a human.

The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of a several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 is a set of images identifying Lutzomyia longipalpis salivary gland anti-complement factors. Fig. IA is a graph demonstrating the ability of various Lu. longipalpis salivary proteins (LuIoSP) to consume complement, as measured by % cell lysis. Fig. IB is a graph showing the inhibition of alternative pathway (AP)- mediated hemolysis of rabbit erythrocytes (E_R) by various LuIoSP. Fig. 1C is a graph demonstrating the inhibition of classical pathway (CP)-mediated hemolysis of antibody-sensitized sheep erythrocytes (EA) by various LuIoSP. Fig. ID is a graph demonstrating the inhibition of C3b deposition on rabbit erythrocytes by various LuIoSP. Fig. IE is a graph showing the measurement of the ability of various LuIoSP to accelerate the decay of the alternative pathway C3/C5 convertase, C3b,Bb.

Figure 2 is a graph demonstrating the inhibition of classical pathway (CP)- mediated hemolysis of antibody-sensitized sheep erythrocytes (EA) by various LuIoSP.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of LJL34.

SEQ ID NO: 2 is the nucleic acid sequence of LJL34.

SEQ ID NO: 3 is the amino acid sequence of LJL18.

SEQ ID NO: 4 is the nucleic acid sequence of LJL18.

SEQ ID NO: 5 is the amino acid sequence of LJS 193.

SEQ ID NO: 6 is the nucleic acid sequence of LJS193.

SEQ ID NO: 7 is the amino acid sequence of LJS201.

SEQ ID NO: 8 is the nucleic acid sequence of LJS201.

SEQ ID NO: 9 is the amino acid sequence of LJLl 3.

SEQ ID NO: 10 is the nucleic acid sequence of LJL13.

SEQ ID NO: 11 is the amino acid sequence of LJL23.

SEQ ID NO: 12 is the nucleic acid sequence of LJL23.

SEQ ID NO: 13 is the amino acid sequence of LJMlO.

SEQ ID NO: 14 is the nucleic acid sequence of LJMlO.

SEQ ID NO: 15 is the amino acid sequence of LJL143.

SEQ ID NO: 16 is the nucleic acid sequence of LJL143.

SEQ ID NO: 17 is the amino acid sequence of LJS 142.

SEQ ID NO: 18 is the nucleic acid sequence of LJS142.

SEQ ID NO: 19 is the amino acid sequence of LJL17.

SEQ ID NO: 20 is the nucleic acid sequence of LJL17.

SEQ ID NO: 21 is the amino acid sequence of LJM06.

SEQ ID NO: 22 is the nucleic acid sequence of LJM06.

SEQ ID NO: 23 is the amino acid sequence of LJM17.

SEQ ID NO: 24 is the nucleic acid sequence of LJM17.

SEQ ID NO: 25 is the amino acid sequence of LJL04.

SEQ ID NO: 26 is the nucleic acid sequence of LJL04.

SEQ ID NO: 27 is the amino acid sequence of LJMl 14.

SEQ ID NO: 28 is the nucleic acid sequence of LJMl 14. SEQ ID NO: 29 is the amino acid sequence of LJMl Il. SEQ ID NO: 30 is the nucleic acid sequence of LJMl 11. SEQ ID NO: 31 is the amino acid sequence of LJM78. SEQ ID NO: 32 is the nucleic acid sequence of LJM78. SEQ ID NO: 33 is the amino acid sequence of LJS238. SEQ ID NO: 34 is the nucleic acid sequence of LJS238. SEQ ID NO: 35 is the amino acid sequence of LJS 169. SEQ ID NO: 36 is the nucleic acid sequence of LJS169. SEQ ID NO: 37 is the amino acid sequence of LJLIl. SEQ ID NO: 38 is the nucleic acid sequence of LJLIl. SEQ ID NO: 39 is the amino acid sequence of LJL08. SEQ ID NO: 40 is the nucleic acid sequence of LJL08. SEQ ID NO: 41 is the amino acid sequence of LJS 105. SEQ ID NO: 42 is the nucleic acid sequence of LJS105. SEQ ID NO: 43 is the amino acid sequence of LJL09. SEQ ID NO: 44 is the nucleic acid sequence of LJL09. SEQ ID NO: 45 is the amino acid sequence of LJL38. SEQ ID NO: 46 is the nucleic acid sequence of LJL38. SEQ ID NO: 47 is the amino acid sequence of LJM04. SEQ ID NO: 48 is the nucleic acid sequence of LJM04. SEQ ID NO: 49 is the amino acid sequence of LJM26. SEQ ID NO: 50 is the nucleic acid sequence of LJM26. SEQ ID NO: 51 is the amino acid sequence of LJS03. SEQ ID NO: 52 is the nucleic acid sequence of LJS03. SEQ ID NO: 53 is the amino acid sequence of LJS 192. SEQ ID NO: 54 is the nucleic acid sequence of LJS192. SEQ ID NO: 55 is the amino acid sequence of LJM19. SEQ ID NO: 56 is the nucleic acid sequence of LJMl 9. SEQ ID NO: 57 is the amino acid sequence of LJL138. SEQ ID NO: 58 is the nucleic acid sequence of LJL138. SEQ ID NO: 59 is the amino acid sequence of LJL15. SEQ ID NO: 60 is the nucleic acid sequence of LJL15. SEQ ID NO: 61 is the amino acid sequence of LJL91. SEQ ID NO: 62 is the nucleic acid sequence of LJL91. SEQ ID NO: 63 is the amino acid sequence of LJMl 1. SEQ ID NO: 64 is the nucleic acid sequence of LJMl 1. SEQ ID NO: 65 is the amino acid sequence of LJS 138. SEQ ID NO: 66 is the nucleic acid sequence of LJS138. SEQ ID NO: 67 is the amino acid sequence of LJL124. SEQ ID NO: 68 is the nucleic acid sequence of LJL124. SEQ ID NO: 69 is the amino acid sequence of LJL35. SEQ ID NO: 70 is the nucleic acid sequence of LJL35. SEQ ID NO: 71 is an oligonucleotide primer.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

/. Abbreviations

AP alternative pathway

CP classical pathway

CVF cobra venom factor

EA antibody-coated sheep erythrocytes

ER rabbit erythrocytes

GVB gelatin/veronal buffered saline

GVB++ GVB, 0.15 mM CaCl₂, 0.5 mM MgCl₂

GVBE GVB, 10 mM EDTA, MgEGTA, 0.1 M MgCl₂, 0.1 M EGTA pH 7.3

LuIoSP Lu. longipalpis salivary proteins

MAC membrane attack complex

NHS normal human serum

RBC red blood cell

TBS tris buffered saline

VBS veronal buffered saline

//. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Amplification of a nucleic acid molecule (for example, a DNA or RNA molecule): A technique that increases the number of copies of a nucleic acid molecule in a specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP 0320308; gap filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBA™ RNA transcription- free amplification, as disclosed in U.S. Patent No. 6,025,134.

Antibody: immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for instance, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (for example, IgG, IgM, IgD) includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term "antibody." Specific, non-limiting examples of binding fragments encompassed within the term antibody include (i) an Fab fragment consisting of the V_L, V_H, CL, and CHl domains; (ii) an Fd fragment consisting of the V_H and CHl domains; (iii) an Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al, Nature 341:544-546, 1989) which consists of a V_H domain; (v) an isolated complimentarity determining region (CDR); and (vi) an F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. Further, non-specific examples include single chain Fv proteins ("scFv") and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (for example, see U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 0256654; EP 0120694; EP 0125023; Faoulkner et al, Nature 298:286, 1982; Morrison, /. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non- human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as dogs.

Complement system (Complement): The complement system is a biochemical cascade of events that supplements a subject's immune system and is therefore a very important first line of defense against pathogens. The complement system consists of a number of small proteins which normally circulate in the plasma in an inactive state. These proteins are cleaved by proteases when the system is activated. The cleaved proteins in turn activate other proteins. This pattern of sequential activation results in an expanding cascade of activity. The complement system can be referred to as "complement." In some circumstances, "complement" refers to less than the entire set of components of the complement system. Activation of the complement system encompasses three different pathways - the classical pathway, the alternative pathway, and the lectin pathway (see Markiewski et ah, Am. J. Path, 171:715-727, 2007, incorporated by reference). Classical pathway components are labeled with a C and a number (e.g. Cl, C3). Alternative pathway components are lettered (e.g. B, P, D). Cleavage fragments are designated with a small letter following the designation of the component (e.g. C3a and C3b are fragments of C3). Inactive C3b is designated iC3b. Polypeptide chains of complement proteins are designated with a Greek letter after the component (e.g., C3 alpha, and C3 beta are the alpha- and beta-chains of C3). Cell membrane receptors for C3 are abbreviated CRl, CR2, CR3, and CR4. The central step of the complement cascade resides in the formation of a C3-convertase, which cleaves C3 to C3b and C3a. Subsequently, the resulting C3b can act as a part of a C5- convertase, which cleaves C5 in C5b and C5a. In the terminal pathway, the gradual accumulation of C6, C7, C8 and several molecules C9 results in the formation of the membrane attack complex (MAC) which is capable of forming a pore in the membrane of the target cells, thereby effecting lysis of the cells.

The classical pathway of the complement system is a major effector of the humoral branch of the human immune response. The trigger activating the classical pathway is either IgG or IgM antibody bound to antigen. Binding of antibody to antigen exposes a site on the antibody which is a binding site for the first complement component, CIq of the Cl-complex (CIq, two molecules of CIr, and two molecules of CIs). Activated Cl cleaves both C4 and C2 into two fragments (C4 into C4a and C4b, and C2 into C2a and C2b). C4b interacts with C2a to form C4b2a, also known as the C3 convertase. C3 convertase converts the next complement component, C3 into C3a and the active form of C3, C3b. Some C3b binds to C4b2a to form C4b2a3b (C5 convertase). C5 convertase catalyzes the cleavage of hundreds of C5 complement component into C5a and C5b. C5b binds to the antigen surface, which is the initial step in the formation of the membrane attack complex (MAC).

Activation of the alternative complement pathway begins when C3b binds to the cell wall and other cell components of the pathogens and/or to IgG antibodies. Factor B then combines with cell-bound C3b and forms C3bB. C3bB is then split into Bb and Ba by Factor D, to form the alternative pathway C3 convertase, C3bBb. Properdin, a serum protein, then binds C3bBb and forms C3bBbP that functions as a C3 convertase, which enzymatically splits C3 molecules into C3a and C3b. At this point, the alternative complement pathway is activated. Some of C3b binds to C3bBb to form C3bBb3b, which is capable of splitting C5 molecules into C5a and C5b.

The lectin pathway begins with the recognition and binding of pathogen- associated molecular patterns by lectin proteins, such as mannose-binding lectin (MBL), and is triggered in the absence of antibody. The lectin pathway requires C2 and C4 complement components for the generation of the C3 convertase and is homologous to the classical pathway, but with opsonin, MBL, and ficolins, instead of CIq. This pathway is activated by binding MBL to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases, MASP-I, and MASP-2 (very similar to CIr and CIs, respectively), which can then split C4 into C4a and C4b and C2 into C2a and C2b. C4b and C2a then bind together to form the C3 -convertase, as in the classical pathway. Ficolins are homologous to MBL and function via MASP in a similar way.

Conservative variants: Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl. Thus, in one embodiment, non-conservative substitutions are those that reduce an activity or antigenicity. cDNA (complementary DNA): A piece of DNA lacking internal, non- coding segments (introns) and expression control sequences. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

Degenerate variant: A polynucleotide encoding a sand fly salivary gland polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the sand fly salivary gland polypeptide encoded by the nucleotide sequence is unchanged.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, for instance, that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. Specific, non-limiting examples of an epitope include a tetra- to penta- peptide sequence in a polypeptide, a tri- to penta-glycoside sequence in a polysaccharide. In the animal most antigens will present several or even many antigenic determinants simultaneously. Such a polypeptide may also be qualified as an immunogenic polypeptide and the epitope may be identified as described further.

Effector molecule (EM): The portion of a chimeric molecule that is intended to have a desired effect on a cell or system or substance to which the chimeric molecule is targeted. The term effector molecule is interchangeable with effector moiety, therapeutic agent, diagnostic agent, and similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins (including monoclonal antibodies and antigen-binding fragments of monoclonal antibodies), peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, recombinant viruses or toxins. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as ³²P, ¹²⁵I, and ¹³¹I, fluorophores, chemiluminescent agents, and enzymes.

Expression Control Sequences: Nucleic acid sequences that control and regulate the expression of a nucleic acid sequence, such as a heterologous nucleic acid sequence, to which it is operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, polyA signals, a start codon (for instance, ATG) in front of a protein-encoding polynucleotide sequence, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription of a nucleic acid. Promoters may be cell-type specific or tissue specific. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see for example, Bitter et al. , Methods in Enzymology 153:516-544, 1987).

For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac-hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells. In one embodiment, the promoter is a cytomegalovirus promoter.

Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Also includes the cells of the subject.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. An immune response can be a cellular response or a humoral response. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). The response can also be a nonspecific response (not targeted specifically to salivary polypeptides) such as production of lymphokines. In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a ThI or a Th2 (subsets of helper T cells) response. In yet another embodiment, the response is a B cell response, and results in the production of specific antibodies.

Immunogenic polypeptide (immunogenic agent): A polypeptide which comprises an allele- specific motif, an epitope or other sequence such that the polypeptide will bind an antibody and induce a humoral response, an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, and/or a B cell response (for example, antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived.

In one embodiment, immunogenic polypeptides are identified using sequence motifs or other methods known in the art. Typically, algorithms are used to determine the "binding threshold" of polypeptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic. The algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif- containing polypeptide. Within the context of an immunogenic polypeptide, a "conserved residue" is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a polypeptide. In one embodiment, a conserved residue is one where the MHC structure may provide a contact point with the immunogenic polypeptide.

Immunogenic composition: A composition that, when administered to a subject induces an immune response to a polypeptide, for example a Lu. longipalpis salivary polypeptide.

Isolated: An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a "labeled polypeptide" refers to incorporation of another molecule in the polypeptide. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as ³⁵S or ¹³¹I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

Leishmaniasis: A parasitic disease spread by the bite of infected sand flies. The trypanosomatid parasite of the genus Leishmania is the etiological agent of a variety of disease manifestations, which are collectively known as leishmaniasis. Leishmaniasis is prevalent through out the tropical and sub-tropical regions of Africa, Asia, the Mediterranean, Southern Europe (old world), and South and Central America (new world). The old world species are transmitted by the sand fly vector Phlebotomus sp. Humans, wild animals and domestic animals (such as dogs) are known to be targets of these sand flies and to act as reservoir hosts or to develop leishmaniasis.

Cutaneous leishmaniasis starts as single or multiple nodules that develop into ulcers in the skin at the site of the bite. The chiclero ulcer typically appears as a notch-like loss of tissue on the ear lobe. The incubation period ranges from days to months, even a year in some cases. The sores usually last months to a few years, with most cases healing on their own. The mucocutaneous type can develop into erosive lesions in the nose, mouth, or throat and can lead to severe disfigurement. Visceral leishmaniasis often has fever occurring in a typical daily pattern, abdominal enlargement with pain, weakness, widespread swelling of lymph nodes, and weight loss, as well as superimposed infections because of a weakened immune system. Visceral leishmaniasis (VL) can result in high death rates. The onset of symptoms can be sudden, but more often tends to be insidious.

Lutzomyia longipalpis (Lu. longipalpis): A species of sand fly endogenous to the New World (South and Central America). This sand fly is the principal vector of American visceral leishmaniasis, a potentially fatal disease that primarily affects children in several countries of South and Central America.

Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells. A lymphocyte can also be referred to as a leukocyte.

Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Oligonucleotide: A linear polynucleotide sequence of up to about 100 nucleotide bases in length.

Open reading frame (ORF): A nucleic acid sequence having a series of nucleotide triplets (codons), starting with a start codon and ending with a stop codon, coding for amino acids without any internal termination codons. These sequences are usually translatable into a polypeptide.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Parenteral: Refers to administration other than through the alimentary canal (the digestive tract), such as by subcutaneous, intramuscular, intrasternal or intravenous administration.

Peptide tag: A peptide sequence that is attached (for instance through genetic engineering) to another peptide or a protein, to provide a function to the resultant fusion. Peptide tags are usually relatively short in comparison to a protein to which they are fused; by way of example, peptide tags are four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids. Usually a peptide tag will be no more than about 100 amino acids in length, and may be no more than about 75, no more than about 50, no more than about 40, or no more than about 30.

Peptide tags confer one or more different functions to a fusion protein (thereby "functionalizing" that protein), and such functions can include antibody binding (an epitope tag), purification, and differentiation (e.g., from a native protein). In addition, a recognition site for a protease, for which a binding antibody is known, can be used as a specifically cleavable epitope tag. The use of such a cleavable tag can provide selective cleavage and activation of a protein (e.g., by replacing the cleavage site in TGF- βl with that for pro-caspase 3).

Detection of the tagged molecule can be achieved using a number of different techniques. These include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("western"), and affinity chromatography.

Epitope tags add a known epitope (antibody binding site) on the subject protein, providing binding of a known and often high-affinity antibody, and thereby allowing one to specifically identify and track the tagged protein that has been added to a living organism or to cultured cells. Examples of epitope tags include the myc, T7, GST, GFP, HA (hemagglutinin) and FLAG tags. The first four examples are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. Patent Nos. 4,703,004 and 4,851,341).

Purification tags are used to permit easy purification of the tagged protein, such as by affinity chromatography. A well-known purification tag is the hexa- histidine (6x His) tag, literally a sequence of six histidine residues. The 6x His protein purification system is available commercially from QIAGEN (Valencia, CA), under the name of QIAexpress®. A single tag peptide can serve more than one purpose; any attached tag, for instance, will increase the molecular weight of the fusion protein and thereby permit differentiation between the tagged and native proteins. Antibodies specific for an "epitope tag" can be used to construct an immunoaffinity column, thus permitting an epitope tag to be used for purification of the tagged protein. Likewise, in some instances monoclonal antibodies specific for a purification tag are available (e.g. anti-6x His peptide monoclonal antibodies, which are available through QIAGEN or CLONTECH, Palo Alto, CA).

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the anticoagulant polypeptides disclosed herein.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Phlebotomus ariasi (P. ariasi): A species of Phlebotomus (sand flies) genus endogenous to the Old World, in particular to southern Europe and Mediterranean countries, more particularly to Spain and France. This sand fly is a proven vector of visceral leishmaniasis. P. ariasi is a member of the subgenera of Phlebotomus Larroussius.

Phlebotomus papatasi (P. papatasi): A species of Phlebotomus (sand flies) genus endogenous to the Old World, in particular to southern Europe, and Mediterranean countries, more particularly to France, Italy, Greece, Morocco, and Spain. This sand fly is a proven vector of the visceral leishmaniasis.

Phlebotomus perniciosus (P. perniciosus): A species of Phlebotomus (sand flies) genus endogenous to the Old World, in particular to southern Europe, and Mediterranean countries, more particularly to France, Italy, Greece, Morocco, and Spain. This sand fly is a proven vector of the visceral leishmaniasis. P. perniciosus is a member of the subgenera of Phlebotomus Larroussius.

Polynucleotide: The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotide at least 10 bases in length, thus including oligonucleotides and genes. A recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA) independent of other sequences. The polynucleotides can be ribonucleotides, deoxyribonucleo tides, or modified forms of either nucleotide. The term includes single -and double -stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length (thus encompassing oligopeptides, peptides, and proteins) or post-translational modification (for example, glycosylation, phosphorylation, or acylation). A polypeptide encompasses also the precursor, as well as the mature protein. In one embodiment, the polypeptide is a polypeptide isolated from Lu. longipalpis, or encoded by a nucleic acid isolated from Lu. longipalpis, such as the Lu. longipalpis polypeptides disclosed herein.

Polypeptide Modifications: Sand fly salivary gland polypeptides include synthetic embodiments of polypeptides described herein. In addition, analogues (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed polypeptide sequences) and variants (homologs) of these proteins can be utilized in the methods described herein. Each polypeptide of the disclosure is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.

Polypeptides may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified polypeptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a Ci-Ci₆ ester, or converted to an amide of formula NR₁R₂ wherein Ri and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric, and other organic salts, or may be modified to Ci-Ci₆ alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chains may be converted to Ci-Ci₆ alkoxy or to a Ci-Ci₆ ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine, or iodine, or with Ci-Ci₆ alkyl, Ci-Ci₆ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C₄ alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of a L. longipalpis polypeptide having measurable or enhanced ability to generate an immune response. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs," Klegerman & Groves (eds.), 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Also included are mimetics prepared using such techniques.

Preventing, treating or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

Probes and primers: A probe comprises an isolated polynucleotide attached to a detectable label or reporter molecule. Primers are short polynucleotides. In one embodiment, polynucleotides are 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise at least 15, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides. Promoter: A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, for example, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see for example, Bitter et al. , Methods in Enzymology 153:516-544, 1987).

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. A polypeptide preparation is substantially purified such that the polypeptide represents several embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, of the total polypeptide content of the preparation. The same applies for polynucleotides. The polypeptides disclosed herein can be purified by any of the means known in the art (see, for example, Guide to Protein Purification, Deutscher (ed.), Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982).

Recombinant: A recombinant polynucleotide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. In one embodiment, a recombinant polynucleotide encodes a fusion protein.

Selectively hybridize: Hybridization under moderately or highly stringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, GC v. AT content), and nucleic acid type (for example, RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.

A specific, non- limiting example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). One of skill in the art can readily determine variations on these conditions (for example, Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et ah, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The hydridization conditions can be carried out over 2 to 16 hours. Washing can be carried out using only one of the above conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.

Selective inhibition of classical but not alternative complement pathway: Inhibition of activation of the complement pathway by an agent, for example a sand fly salivary gland anti-complement polypeptide or a polynucleotide encoding such a polypeptide, whereas alternative pathway activation is substantially not affected. Activity of one or more components of the classical complement pathway is inhibited (substantially decreased), whereas the activity of the components of the alternative pathway are substantially not decreased. In addition, activity of the components of the lectin pathway may not be substantially decreased. A decrease in the percent lysis of antibody-coated sheep erythrocytes in the presence of an agent, compared to the percent lysis of antibody-coated sheep erythrocytes in the absence of the agent, is a measure of the inhibitory activity of the agent on the classical pathway of complement.

Sequence identity: The similarity between amino acid sequences is expressed in terms of the percentage identity between the sequences. The higher the percentage, the more similar the two sequences are. Homologs or variants of a sand fly salivary gland polypeptide will possess a relatively significant high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, /. MoI. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. ScL USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al , Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. ScL USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994 presents a detailed consideration of sequence alignment methods and identity calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. MoI. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Homologs and variants of a sand fly salivary gland polypeptide are typically characterized by possession of at least 75%, for example at least 80%, sequence identity counted over the full length alignment with the amino acid sequence of the Lu. longipalpis polypeptide using the NCBI Blast 2.0, gapped blastp set to default parameters. The comparison between the sequences is made over the full length alignment with the amino acid sequence given in this present disclosure, employing the Blast 2 sequences function using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).

When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologues and, variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologues could be obtained that fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to a defined target. Thus, for example, a Lu. longipalpis specific binding agent is an agent that binds substantially to a Lu. longipalpis polypeptide. In one embodiment, the specific binding agent is a monoclonal or polyclonal antibody that specifically binds the Lu. longipalpis polypeptide.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human veterinary subjects, including human and non-human mammals. In one embodiment, the subject is a member of the canine family, such as a dog. In another embodiment, the subject is a human.

T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. ThI and Th2 cells are functional subsets of helper T cells. ThI cells secrete a set of cytokines, including interferon-gamma, and whose principal function is to stimulate phagocyte-mediated defense against infections, especially related to intracellular microbes. Th2 cells secrete a set of cytokines, including interleukin (IL)-4 and IL-5, and whose principal functions are to stimulate IgE and eosinophil/mast cell-mediated immune reactions and to downregulate ThI responses.

CD8⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, a CD8 T cells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell is a suppressor T cell.

Therapeutically active polypeptide: An agent, such as a sand fly salivary gland polypeptide, that inhibits the complement system (inhibits complement activation) by inhibiting the activity of a component of the complement system, as measured by a clinical response, for example. The therapeutically active polypeptide results in the inhibition of a component of the complement system, or a measurable reduction in symptoms related to pathologies and diseases believed to have a complement-mediated component. The therapeutically active polypeptide can also be used to prevent or treat a subject against visceral leishmaniasis transmitted by sand flies. Therapeutically active molecules can also be made from nucleic acids. Examples of a nucleic acid based therapeutically active molecule is a nucleic acid sequence that encodes a sand fly salivary gland polypeptide, wherein the nucleic acid sequence is operably linked to a control element such as a promoter. Therapeutically active agents can also include organic or other chemical compounds that mimic the effects of the sand fly salivary gland polypeptide.

The terms "therapeutically effective fragment of a sand fly salivary gland polypeptide" includes any fragment of the sand fly salivary gland polypeptide, or variant of the sand fly salivary gland polypeptide, or fusion protein including a sand fly salivary gland polypeptide, that retains a function of the sand fly salivary gland polypeptide, or retains the ability to reduce the symptoms related to a disorder associated with complement activation.

Thus, in one embodiment, a therapeutically effective amount of a fragment of sand fly salivary gland polypeptide is an amount used to inhibit the activity of a component of the complement system. In yet another embodiment, a therapeutically effective amount of a fragment of a sand fly salivary gland polypeptide is an amount of use to prevent or treat a disorder associated with complement activity in a subject. Specific, non-limiting examples of a polypeptide fragment are the N-terminal half or the C-terminal half of one of the sand fly salivary gland polypeptide disclosed herein. It should be noted that fusion proteins are included, such as a fusion with six histidine residues, a c-myc tag, or any other polypeptide tag. Such fusions are known to one of skill in the art, and are often used in protein purification.

Transduced: A transduced cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transduction encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Transmission-blocking vaccine: Compositions containing arthropod vector-specific or parasite-specific polypeptides which are administered to a subject, giving rise to an immune response against the polypeptide. The resultant antibodies can block transmission of a parasite from the subject to the arthropod vector, preventing the parasite from completing its life cycle. The antibodies also can be ingested by the arthropod with a blood meal, which can prevent parasite development in the arthropod, thereby blocking transmission of the parasite from the arthropod vector to a human or animal. An amount of prophylactic composition sufficient to result in blocking of transmission is defined to be an immunologically effective dose.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transduced host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Hence "comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All sequence database references are incorporated by reference as of the date of the filing of this application. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Anti-Complement Compositions

The major functions of the complement system (complement) in innate immunity and specific humoral immunity are to promote phagocytosis of pathogens on which complement is activated, stimulate inflammation, and induce the lysis of these pathogens. One example of the importance of complement is host defense against bacteria with polysaccharide-rich capsules, such a pneumococci, which is mediated entirely by humoral immunity. IgM antibodies against the capsular polysaccharides bind to the bacteria, activate the classical pathway of complement, and cause phagocytotic clearance of the bacteria. Complement-mediated lysis of pathogens is mediated by the membrane attack complex (MAC), particularly for bacteria of the genus Neisseria. Another example of the function of complement is the ability of proteolytic complement fragments C5a, C4a, and C3a (anaphylatoxins and potent inflammatory mediators) to induce an acute inflammatory response by acting on mast cells and basophils, causing degranulation and the release of histamines and other active peptides. These peptides increase the permeability of the vascular walls of microbes allowing neutrophils to migrate to the area and phagocytose the pathogen. In another aspect of the complement system, certain components, most notably C4b and C5b, act as opsonins. Many phagocytic cells have receptors for these complement products. Antigens coated with either of these molecules are opsonized and are more likely to be ingested by phagocytes.

The complement system has the potential to be extremely damaging to host tissues, therefore its activation must be tightly regulated. The complement system is regulated by complement control proteins, which are present at a higher concentration in the blood plasma than the complement proteins themselves. As deficiencies of any of the protein components may lead to abnormal or undesirable patterns of complement activation, the complement system is also involved in human disease. For example, if regulatory components are absent, or a component of the complement system has increased or abnormal activity, too much complement activation (increased or abnormal complement activity) may occur leading to excess inflammation, cell lysis, and damage to host cells. In addition, the complement system may be undesirably activated in response to abnormal stimuli, such as persistent microbes or antibodies against self antigens, thereby significantly contributing to the pathology of the disease. Thus, anti-complement polypeptides can be used to inhibit excess, undesirable, or abnormal activation of components of the complement system. In contrast, subjects who are deficient in or have a total lack of one or more complement components, for example C3, are extremely susceptible to lethal bacterial infections.

The sand fly salivary gland polypeptides disclosed herein exhibit anti- complement activity. A polypeptide with anti-complement activity (referred to herein as an anti-complement polypeptide) can be a polypeptide isolated from the salivary gland or saliva of any sand fly, for example (but not limited to) Phlebotomus papatasi, Phlebotomus ariasi, Phlebotomus perniciosus, Lutzomyia longipalpis, Phlebotomus argentipes, Phlebotomus orientalis, Phlebotomus duboscqi, Phlebotomus arabicus, Lutzomyia intermedia, Lutzomyia shanoni, or Lutzomyia whitmani. Examples of these polypeptide sequences are disclosed in PCT/US2003/034453 filed October 29, 2003; PCT Application No. PCT/US2003/029833, filed September 18, 2003; and PCT/US02/19663, filed June 18, 2002, which are incorporated herein by reference. A particular Lutzomyia longipalpis (Lu. longipalpis) salivary polypeptide (residues 23-115 of SEQ ID NO: 55; also referred to as the mature form of LJM19) exhibited surprisingly potent anti- complement activity via the classical pathway, when compared to other Lu. longipalpis salivary gland polypeptides. For example, residues 23-115 of SEQ ID NO: 55 consistently exhibited a substantially complete (approximately 100%, for example 99%, 98%, 97%) inhibition of complement activity, as demonstrated by a substantial lack (approximately 0%) of classical pathway-mediated cell lysis, whereas other Lu. longipalpis salivary gland polypeptides had no measurable effect (or minimal effect) on complement activity when tested under the same conditions. Residues 23-115 of SEQ ID NO: 55 had substantially no effect on alternative pathway-mediated cell lysis. Thus, this polypeptide acts specifically in selectively blocking the classical pathway, as compared to the alternative pathway. In some embodiments, other Lu. longipalpis salivary gland polypeptides (residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO: 49, and residues 19-399 of SEQ ID NO: 63; also referred to as LJM04, LJM26, or LJMI l, respectively) demonstrate an inhibition of classical pathway- mediated cell lysis, alternative pathway-mediated cell lysis, or both.

The sand fly salivary gland polypeptides disclosed herein have an inhibitory effect on the complement system (an inhibition or reduction of complement activity), thereby inhibiting or reducing the lytic activity of the complement system. The complement system (complement) includes the components of the classical pathway, the alternative pathway, and the lectin pathway. Thus, the sand fly salivary gland polypeptides disclosed herein have an inhibitory effect on one or more components of the complement system, thereby inhibiting or reducing the lytic activity of the complement system. An inhibition (or reduction) of complement activity can be measured by any method known to one of skill in the art. In one embodiment, a decrease in cell lysis (for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% decrease in cell lysis), as measured by a hemolytic assay, when a sample is contacted with one or more components of the complement system and a sand fly salivary gland polypeptide, is an indication that the polypeptide has anti-complement activity. In some embodiments, the reduction or inhibition of complement activity is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In yet other embodiments, an inhibition or reduction in complement activity is measured by a clinical response in vivo, such as a measurable reduction in symptoms related to various pathologies or syndromes, such as asthma, lupus erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, and ischemia-reperfusion injuries.

In some embodiments, a sand fly salivary gland polypeptide disclosed herein has an inhibitory effect on a component of the classical pathway of complement. In other embodiments, a sand fly salivary gland polypeptide disclosed herein has an inhibitory effect on a component of the alternative pathway or a component of the lectin pathway. A specific, non-limiting example of a sand fly salivary gland anti- complement polypeptide, having a surprisingly potent inhibitory effect on a component of the classical pathway, includes the Lu. longipalpis polypeptide having an amino acid sequence (without a signal sequence) set forth as residues 23-115 of SEQ ID NO: 55 (LJM19), or variants or fragments thereof. A specific, non-limiting example of a sand fly salivary gland anti-complement polypeptide, having a potent inhibitory effect on a component of the alternative pathway, includes the Lu. longipalpis polypeptide having an amino acid sequence (without a signal sequence) set forth as residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, or residues 19-399 of SEQ ID NO: 63 (also referred to as LJM04, LJM26, or LJMIl, respectively), or variants or fragments thereof. In yet other non- limiting embodiments, residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63 inhibit the lectin pathway of the complement cascade.

Specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence including a signal sequence, as set forth as SEQ ID NO: 47 (residues 1-139 of SEQ ID NO: 47), SEQ ID NO: 49 (residues 1-446 of SEQ ID NO: 49), SEQ ID NO: 55 (residues 1-115 of SEQ ID NO: 55), or SEQ ID NO: 63 (residues 1-399 of SEQ ID NO: 63), or variants or fragments thereof.

Specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 1-50, 51-75, 76-100, 101-125, 126-139, 21-50, 21-75, 21- 100, 21-125, 21-139, 50-139, 75-139, 100-139, 125-139 of SEQ ID NO: 47, or variants or fragments thereof. Other specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 22-139, 23-239, 24-139, 25- 139, 26-139, 27-139, 28-139, 29-139, 30-139, 31-139, etc of SEQ ID NO: 47, or variants or fragments thereof.

Specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 1-50, 51-75, 76-100, 101-125, 126-150, 151-175, 176-200, 201-225, 226-250, 251-275, 276-300, 301-325, 326-350, 351-375, 376-400, 401- 425, 426-446, 18-50, 18-75, 18-100, 18-125, 18-150, 18-175, 18-200, 18-225, 18- 250, 18-275, 18-300, 18-325, 18-350, 18-375, 18-400, 18-425, 18-446, 50-446, 75- 446, 100-446, 125-446, 150-446, 175-446, 200-446, 225-446, 250-446, 275-446, 300-446, 325-446, 350-446, 375-446, 400-446, 425-446 of SEQ ID NO: 49, or variants or fragments thereof. Other specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 19-446, 20-446, 21-446, 22- 446, 23-446, 24-446, 25-446, 26-446, 27-446, 28-446, etc of SEQ ID NO: 49, or variants or fragments thereof.

Specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 1-50, 51-75, 76-100, 101-115, 23-50, 23-75, 23-100, 23-115, 50-115, 75-115, 100-115, 125-115 of SEQ ID NO: 55, or variants or fragments thereof. Other specific, non-limiting examples of sand fly salivary gland anti- complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 24-115, 25-115, 26-115, 27-115, 28-115, 29- 115, 30-115, 31-115, 32-115, 33-115, etc of SEQ ID NO: 55, or variants or fragments thereof.

Specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 1-50, 51-75, 76-100, 101-125, 126-150, 151-175, 176-200, 201-225, 226-250, 251-275, 276-300, 301-325, 326-350, 351-375, 376-399, 19-50, 19-75, 19-100, 19-125, 19-150, 19-175, 19-200, 19-225, 19-250, 19-275, 19-300, 19-325, 19-350, 19-375, 19-399, 50-399, 75-399, 100-399, 125-399, 150-399, 175- 399, 200-399, 225-399, 250-399, 275-399, 300-399, 325-399, 350-399, 375-399 of SEQ ID NO: 63, or variants or fragments thereof. Other specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues 20- 399, 21-399, 22-399, 23-399, 24-399, 25-399, 26-399, 27-399, 28-399, 29-399, etc of SEQ ID NO: 63, or variants or fragments thereof.

In specific embodiments, sand fly salivary gland anti-complement polypeptides, for example residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or variants or fragments thereof, bind a complement molecule, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5 with very high affinity. In further examples, residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, or residues 19-399 of SEQ ID NO: 63 modulate the activity of a complement molecule, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5. For example, the activity of a complement molecule can be decreased or increased as a result of interaction with the salivary gland polypeptides disclosed herein. In one specific, non- limiting example, the inhibition of C3b activity inhibits the formation of C5 convertase and prevents the conversion of C5 complement component into C5a and C5b.

In one embodiment, the anti-complement polypeptides disclosed herein include homologous polypeptides having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the anti-complement polypeptide. One specific, non- limiting example of a longer homologous sequence that contains residues 21-139 of SEQ ID NO: 47 and has anti-complement activity is the full- length polypeptide set forth as SEQ ID NO: 47 (residues 1-139 of SEQ ID NO: 47). Another specific, non-limiting example of a longer homologous sequence that contains residues 18-446 of SEQ ID NO: 49 and has anti-complement activity is the full-length polypeptide set forth as SEQ ID NO: 49 (residues 1-446 of SEQ ID NO: 49). Another specific, non-limiting example of a longer homologous sequence that contains residues 23-115 of SEQ ID NO: 55 and has anti-complement activity is the full-length polypeptide set forth as SEQ ID NO: 55 (residues 1-115 of SEQ ID NO: 55). Another specific, non-limiting example of a longer homologous sequence that contains residues 19-399 of SEQ ID NO: 63 and has anti-complement activity is the full-length polypeptide set forth as SEQ ID NO: 63 (residues 1-399 of SEQ ID NO: 63).

Yet other specific non-limiting examples of longer homologous sequences include Lu. longipalpis polypeptides having an amino acid sequence set forth as residues 2-139, 3-139, 4-139, 5-139, 6-139, 7-139, 8-139, 9-139, 10-138, etc of SEQ ID NO: 47, or variants or fragments thereof; residues 2-446, 3-446, 4-446, 5-446, 6- 446, 7-446, 8-446, 9-446, 10-446, etc of SEQ ID NO: 49, or variants or fragments thereof; residues 2-115, 3-115, 4-115, 5-115, 6-115, 7-115, 8-115, 9-115, 10-115, etc of SEQ ID NO: 55, or variants or fragments thereof; or residues 2-399, 3-399, 4-399, 5-399, 6-399, 7-399, 8-399, 9-399, 10-399, etc of SEQ ID NO: 63, or variants or fragments thereof.

Fusion proteins including an anti-complement polypeptide can also be produced using methods known to one of skill in the art. In one embodiment, a fusion protein includes an amino acid sequence set forth as residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or conservative variants or fragments thereof, and a marker polypeptide. Fusion proteins, which include the anti-complement polypeptide and retain the anti-complement activity, are also disclosed herein. Such fusion proteins can include, in addition to the anti-complement polypeptide, an effector molecule (such as a monoclonal antibody), a label (such as enzymatic labels, polypeptide epitopes, or fluorescent proteins), or a peptide tag (such as peptide tags that are four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids). Homologs, fragments, variants, and fusion proteins including the anti-complement polypeptides exhibit substantially the same activity as the anti- complement polypeptides. Thus, homologs, fragments, variants, and fusion proteins including the anti-complement polypeptides inhibit or reduce complement activity, as demonstrated by, for example, a reduction or inhibition in cell lysis of a sample. In some embodiments, homologs, fragments, variants, and fusion proteins including the anti-complement polypeptides, inhibit or reduce complement activity by having an inhibitory effect on one or more components of the classical pathway, the alternative pathway, and/or the lectin pathway of complement, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5. In other embodiments, homologs, fragments, variants, and fusion proteins including the anti-complement polypeptides, bind to C3b, or inhibit or reduce C3b activity.

Fragments and variants of the anti-complement polypeptides identified above are disclosed herein and can readily be prepared by one of skill in the art using molecular techniques. In one embodiment, a fragment of an anti-complement polypeptide includes at least 8, 10, 15, 19, 20 23, 25, or 30 amino acids of an anti- complement polypeptide. In some embodiments, fragments of the anti-complement polypeptide include the disclosed amino acid sequence having truncations or internal deletions. The truncations or internal deletions can include at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or more amino acids. In other embodiments, a fragment of an anticoagulant polypeptide includes the N-terminal half or the C-terminal half of the polypeptide. In another embodiment, a fragment of an anti-complement polypeptide includes a specific antigenic epitope found on a full-length anti-complement polypeptide.

One skilled in the art, given the disclosure herein, can purify an anti- complement polypeptide using standard techniques for protein purification and as described herein. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the anti-complement polypeptide can also be determined by amino-terminal amino acid sequence analysis.

Minor modifications of the anti-complement polypeptide primary amino acid sequences may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein.

In another embodiment, the anti-complement composition includes an isolated polynucleotide having a nucleic acid sequence encoding the sand fly salivary gland polypeptides disclosed herein. The polynucleotide can be from any sand fly, for example (but not limited to) Phlebotomus papatasi, Phlebotomus ariasi, Phlebotomus perniciosus, or Lutzomyia longipalpis. Examples of these polynucleotide sequences are disclosed in PCT/US2003/034453 filed October 29, 2003; PCT Application No. PCT/US2003/029833, filed September 18, 2003; and PCT/US02/19663, filed June 18, 2002, which are incorporated herein by reference.

Encompassed by this disclosure are polynucleotides encoding an anti- complement salivary polypeptide such as residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63. Specific, non-limiting examples of an anti-complement nucleic acid sequence include residues 100-456 of SEQ ID NO: 48, residues 147- 1616 of SEQ ID NO; 50, residues 82-360 of SEQ ID NO: 56, or residues 74-1216 of SEQ ID NO: 64, or a degenerate variant thereof. Other specific, non-limiting examples of an anti-complement nucleic acid sequence include residues 40-456 of SEQ ID NO: 48, residues 96-1616 of SEQ ID NO; 50, residues 16-360 of SEQ ID NO: 56, or residues 20-1216 of SEQ ID NO: 64, or a degenerate variant thereof. These polynucleotides include DNA, cDNA, and RNA sequences that encode an anti-complement polypeptide. It is understood that all polynucleotides encoding an anti-complement polypeptide are also included herein, as long as they encode a polypeptide with the recognized activity, such as inhibiting or reducing complement activation, inhibiting or reducing the lytic activity of the complement system, or inhibiting or reducing the activity of a component of the complement system, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5. As used herein, the phrase "nucleic acid molecules encoding the anti-complement polypeptide" includes both nucleic acid molecules encoding the anti-complement polypeptide with the signal sequence and nucleic acid molecules encoding the anti-complement polypeptide without the signal sequence.

The polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the anti- complement polypeptide encoded by the nucleotide sequence is functionally unchanged. The nucleic acid sequences can also include 5' and 3' expression control sequences, such as a start codon (ATG), a stop codon, and a poly A signal.

Also included are fragments of the above-described nucleic acid sequences that are at least 33 bases, at least 36 bases, at least 42 bases or at least 48 bases in length, which is sufficient to permit the fragment to selectively hybridize to a polynucleotide that encodes a disclosed anti-complement polypeptide under specified conditions. In some embodiments, fragments of the nucleic acid sequences include truncations or internal deletions. The truncations or internal deletions can include at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or more nucleic acids. In other embodiments, a fragment of an anticoagulant polynucleotide includes the N-terminal half or the C-terminal half of the sequence encoding the polypeptide. The term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions, which excludes non-related nucleotide sequences.

Another specific non-limiting example of a polynucleotide encoding an anti- complement polypeptide is a polynucleotide having at least 75%, 85%, 90%, 95%, or 99% homology to a nucleotide sequence that encodes a polypeptide having an antigenic epitope or function of an anti-complement polypeptide. Yet another specific non-limiting example of a polynucleotide encoding an anti-complement polypeptide is a polynucleotide that encodes a polypeptide that is specifically bound by an antibody that specifically binds the anti-complement polypeptide.

One specific, non-limiting example of a longer homologous sequence that contains residues 100-456 of SEQ ID NO: 48 and encodes a polypeptide that has anti-complement activity are residues 40-546 of SEQ ID NO: 48, which codes for the full-length polypeptide. Another specific, non- limiting example of a longer homologous sequence is the full-length nucleic acid sequence set forth as SEQ ID NO: 48 (residues 1-456 of SEQ ID NO: 48). Other specific, non-limiting examples of a longer homologous sequence that contains residues 100-456 of SEQ ID NO: 48 and encodes for a polypeptide having anti-complement activity includes polynucleotides having an amino acid sequence set forth as residues 2-456, 3-456, 4- 456, 5-456, 6-456, 7-456, 8-456, 9-456, 10-456 etc, of SEQ ID NO: 48, or degenerate variants thereof.

One specific, non-limiting example of a longer homologous sequence that contains residues 147-1616 of SEQ ID NO: 50 and encodes a polypeptide that has anti-complement activity is residues 96-1616 of SEQ ID NO: 50, which codes for the full-length polypeptide. Another specific, non-limiting example of a longer homologous sequence is the full-length nucleic acid sequence set forth as SEQ ID NO: 50 (residues 1-1616 of SEQ ID NO: 50). Other specific, non-limiting examples of a longer homologous sequence that contains residues 147-1616 of SEQ ID NO: 50 and encodes for a polypeptide having anti-complement activity includes polynucleotides having an amino acid sequence set forth as residues 2-1616, 3-1616, 4-1616, 5-1616, 6-1616, 7-1616, 8-1616, 9-1616, 10-1616 etc, of SEQ ID NO: 50, or degenerate variants thereof.

One specific, non-limiting example of a longer homologous sequence that contains residues 82-360 of SEQ ID NO: 56 and encodes a polypeptide that has anti- complement activity is residues 16-360 of SEQ ID NO: 56, which codes for the full- length polypeptide. Another specific, non-limiting example of a longer homologous sequence is the full-length nucleic acid sequence set forth as SEQ ID NO: 56 (residues 1-360 of SEQ ID NO: 56). Other specific, non-limiting examples of a longer homologous sequence that contains residues 82-360 of SEQ ID NO: 56 and encodes for a polypeptide having anti-complement activity includes polynucleotides having an amino acid sequence set forth as residues 2-360, 3-360, 4-360, 5-360, 6- 360, 7-360, 8-360, 9-360, 10-360 etc, of SEQ ID NO: 56, or degenerate variants thereof.

One specific, non-limiting example of a longer homologous sequence that contains residues 74-1216 of SEQ ID NO: 64 and encodes a polypeptide that has anti-complement activity is residues 20-1216 of SEQ ID NO: 64, which codes for the full-length polypeptide. Another specific, non- limiting example of a longer homologous sequence is the full-length nucleic acid sequence set forth as SEQ ID NO: 64 (residues 1-1216 of SEQ ID NO: 64). Other specific, non-limiting examples of a longer homologous sequence that contains residues 74-1216 of SEQ ID NO: 64 and encodes for a polypeptide having anti-complement activity includes polynucleotides having an amino acid sequence set forth as residues 2-1216, 3-1216, 4-1216, 5-1216, 6-1216, 7-1216, 8-1216, 9-1216, 10-1216 etc, of SEQ ID NO: 64, or degenerate variants thereof.

The anti-complement polynucleotides include a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleo tides, or modified forms of either nucleotide. The term includes single and double forms of either nucleotide.

Recombinant vectors are also disclosed herein that include a polynucleotide encoding a polypeptide or a fragment thereof according to the disclosure. Recombinant vectors include plasmids and viral vectors and may be used for in vitro or in vivo expression.

A plasmid may include a DNA transcription unit, for instance a nucleic acid sequence that permits it to replicate in a host cell, such as an origin of replication (prokaryotic or eukaryotic). A plasmid may also include one or more selectable marker genes and other genetic elements known in the art. Circular and linear forms of plasmids are encompassed in the present disclosure.

For in vivo expression, the promoter is generally of viral or cellular origin. In one embodiment, the cytomegalovirus (CMV) early promoter (CMV-IE promoter), including the promoter and enhancer, is of use. The CMV-IE promoter can be of human or murine origin, or of other origin such as rat or guinea pig (see EP 0260148; EP 0323597; WO 89/01036; Pasleau et al, Gene 38:227-232, 1985; Boshart M. et al, Cell 41:521-530, 1985). Functional fragments of the CMV-IE promoter may also be used (WO 98/00166). The SV40 virus early or late promoter and the Rous Sarcoma virus LTR promoter are also of use. Other promoters include but are not limited to, a promoter of a cytoskeleton gene, such as (but not limited to) the desmin promoter (Kwissa M. et al, Vaccine 18(22):2337-2344, 2000), or the actin promoter (Miyazaki J. et al, Gene 79(2):269-277, 1989). Either constitutive or inducible promoters can be used. When several genes are present in the same plasmid, they may be provided in the same transcription unit or in different units.

The plasmids may also comprise other transcription regulating elements such as, for example, stabilizing sequences of the intron type. In several embodiments the plasmids include the first intron of CMV-IE (Published PCT Application No. WO 89/01036), the intron II of the rabbit β-globin gene (van Ooyen et al. , Science 206:337-344, 1979), the signal sequence of the protein encoded by the tissue plasminogen activator (tPA; Montgomery et al , Cell. MoI. Biol. 43:285-292, 1997), and/or a polyadenylation signal (poly A), in particular the polyA of the bovine growth hormone (bGH) gene (U.S. Patent No. 5,122,458) or the polyA of the rabbit β- globin gene or of SV40 virus.

In a specific, non-limiting example, the pVR1020 plasmid (VICAL Inc.; Luke C. et al, Journal of Infectious Diseases 175:91-97, 1997; Hartikka J. et al, Human Gene Therapy 7:1205-1217, 1996)) can be utilized as a vector for the insertion of such a polynucleotide sequence, generating recombinant plasmids.

Various viral vectors are also of use with a polynucleotide encoding an anti- complement polypeptide. A specific, non-limiting example includes recombinant poxvirus, including avipox viruses, such as the canarypox virus. Another specific, non-limiting example includes recombinant poxvirus, including vaccinia viruses (U.S. Patent No. 4,603,112), such as attenuated vaccinia virus such as NYVAC (see U.S. Patent No. 5,494,807) or Modified Vaccinia virus Ankara (MVA, Stickl H. and Hochstein-Mintzel V., Munch. Med. Wschr. 113:1149-1153, 1971; Sutter G. et al, Proc. Natl. Acad. ScL USA 89: 10847-10851, 1992; Carroll M. W. et al, Vaccine 15(4):387-394, 1997; Stittelaar K. J. et al, J. Virol. 74(9):4236-4243, 2000; Sutter G. et al, Vaccine 12(l l):1032-1040, 1994). When avipox viruses are used, canarypox viruses (U.S. Patent No. 5,756,103) and fowlpox viruses (U.S. Patent No. 5,766,599) are of use, such as attenuated viruses. When the expression vector is a poxvirus, the heterologous polynucleotide can be inserted under the control of a poxvirus specific promoter, such as the vaccinia virus 7.5kDa promoter (Cochran et al, J. Virology 54:30-35, 1985), the vaccinia virus I3L promoter (Riviere et al, J. Virology 66:3424-3434, 1992), the vaccinia virus HA promoter (Shida, Virology 150:451-457, 1986), the cowpox virus ATI promoter (Funahashi et al, J. Gen. Virol. 69:35-47, 1988), other vaccinia virus H6 promoter (Taylor et al, Vaccine 6:504-508, 1988; Guo et al, J. Virol. 63:4189-4198, 1989; Perkus et al, J. Virol. 63:3829-3836, 1989).

Other viral vectors of use are herpes virus or adenovirus vectors. Specific, non-limiting examples include a canine herpes virus (CHV) or canine adenovirus (CAV) vector (for example, see U.S. Patent No. 5,529,780; U.S. Patent No. 5,688,920; Published PCT Application No. WO 95/14102). For CHV, the insertion sites may be in particular in the thymidine kinase gene, in the ORF3, or in the UL43 ORF (see U.S. Patent No. 6,159,477). For CAV, the insertion sites may be in particular in the E3 region or in the region located between the E4 region and the right ITR region (see U.S. Patent No. 6,090,393; U.S. Patent No. 6,156,567). In one embodiment in CHV or CAV vectors the insert is in general under the control of a promoter (as described above for the plasmids), such as CMV-IE promoter.

Multiple insertions can be done in the same vector using different insertion sites or using the same insertion site. When the same insertion site is used, each polynucleotide insert is inserted under the control of different promoters. The insertion can be done tail-to-tail, head-to-head, tail-to-head, or head-to-tail. IRES elements (Internal Ribosome Entry Site, see European Patent EP 0803573) can also be used to separate and to express multiple inserts operably linked to the same promoter. Bacterial vectors can also be used for in vivo expression.

Any polynucleotide according to the disclosure can be expressed in vitro by DNA transfer or expression vectors into a suitable host cell. The host cell may be prokaryotic or eukaryotic. The term "host cell" also includes any progeny of the subject host cell. Methods of stable transfer, meaning that the foreign polynucleotide is continuously maintained in the host cell, are known in the art. Host cells can include bacteria (for example, Escherichia coli), yeast, insect cells, and vertebrate cells. Methods of expressing DNA sequences in eukaryotic cells are well known in the art.

As a method for in vitro expression, recombinant Baculovirus vectors (for example, Autographa California Nuclear Polyhedrosis Virus (AcNPV)) can be used with the nucleic acids disclosed herein. For example, polyhedrin promoters can be utilized with insect cells (for example, Spodoptera frugiperda cells, like Sf9 cells available at the ATCC under the Accession number CRL-1711, or Sf21 cells) (see for example, Smith et al, MoI. Cell Biol. 3:2156-2165, 1983; Pennock et al., Mol. Cell Biol. 4: 399-406, 1994; Vialard et al., J. Virol. 64:37-50, 1990; Verne A., Virology 167:56-71, 1988; O'Reilly et al., "Baculovirus expression vectors, A laboratory manual," New York Oxford, Oxford University Press, 1994; Kidd I. M. & Emery V.C., "The use of baculoviruses as expression vectors," Applied Biochemistry and Biotechnology 42:37-159, 1993; European Patent No. EP 0370573; European Patent No. EP 0265785; U.S. Patent No. 4,745,051). For expression the BaculoGold ™ Starter Package (Cat # 21001K) from Pharmingen (Becton Dickinson) can be used.

As a method for in vitro expression, recombinant E. coli can be used with a vector. For example, when cloning in bacterial systems, inducible promoters such as arabinose promoter, pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter), and the like may be used.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transduction of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with anti- complement polynucleotide sequences, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector (see above), such as a herpes virus or adenovirus (for example, canine adenovirus T), to transiently transduce eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). In addition, a transfection agent can be utilized, such as dioleoyl-phosphatidyl-ethanolamine (DOPE).

Isolation and purification of recombinantly expressed polypeptide may be carried out by conventional means including preparative chromatography (for example, size exclusion, ion exchange, affinity), selective precipitation and ultrafiltration. Such a recombinantly expressed polypeptide is part of the present disclosure. The methods for production of such a polypeptide are also encompassed, in particular the use of a recombinant expression vector comprising a polynucleotide according to the disclosure and of a host cell.

Any of the sand fly polypeptides disclosed herein can have anti-complement activity. Specific, non-limiting examples of sand fly salivary gland polypeptides, and the nucleic acid sequences encoding them, include those derived from Lu. Longipalpis, as follows:

The LJL34 unprocessed protein (SEQ ID NO: 1) is encoded by nucleic acid residues 30-842 of SEQ ID NO: 2, and the mature protein (amino acid residues 20- 271 of SEQ ID NO: 1) is encoded by nucleic acid residues 87-842 of SEQ ID NO: 2.

The LJL18 unprocessed protein (SEQ ID NO: 3) is encoded by nucleic acid residues 56-532 of SEQ ID NO: 4, and the mature protein (amino acid residues 20- 159 of SEQ ID NO: 3) is encoded by nucleic acid residues 113-532 of SEQ ID NO: 4.

The LJS 193 unprocessed protein (SEQ ID NO: 5) is encoded by nucleic acid residues 216-502 of SEQ ID NO: 6, and the mature protein (amino acid residues 21- 304 of SEQ ID NO: 5) is encoded by nucleic acid residues 276-502 of SEQ ID NO: 6.

The LJS201 unprocessed protein (SEQ ID NO: 7) is encoded by nucleic acid residues 48-353 of SEQ ID NO: 8, and the mature protein (amino acid residues 24- 102 of SEQ ID NO: 7) is encoded by nucleic acid residues 117-352 of SEQ ID NO: 8.

The LJL13 unprocessed protein (SEQ ID NO: 9) is encoded by nucleic acid residues 26-766 of SEQ ID NO: 10, and the mature protein (amino acid residues 20- 247 of SEQ ID NO: 9) is encoded by nucleic acid residues 83-766 of SEQ ID NO: 10.

The LJL23 unprocessed protein (SEQ ID NO: 11) is encoded by nucleic acid residues 18-992 of SEQ ID NO: 12, and the mature protein (amino acid residues 22- 325 of SEQ ID NO: 11) is encoded by nucleic acid residues 81-992 of SEQ ID NO: 12.

The LJMlO unprocessed protein (SEQ ID NO: 13) is encoded by nucleic acid residues 92-571 of SEQ ID NO: 14, and the mature protein (amino acid residues 20- 160 of SEQ ID NO: 13) is encoded by nucleic acid residues 149-571 of SEQ ID NO: 14.

The LJL143 unprocessed protein (SEQ ID NO: 15) is encoded by nucleic acid residues 46-948 of SEQ ID NO: 16, and the mature protein (amino acid residues 24-301 of SEQ ID NO: 15) is encoded by nucleic acid residues 115-948 of SEQ ID NO: 16.

The LJS 142 unprocessed protein (SEQ ID NO: 17) is encoded by nucleic acid residues 25-507 of SEQ ID NO: 18, and the mature protein (amino acid residues 21-161 of SEQ ID NO: 17) is encoded by nucleic acid residues 85-507 of SEQ ID NO: 18.

The LJL17 unprocessed protein (SEQ ID NO: 19) is encoded by nucleic acid residues 28-342 of SEQ ID NO: 20, and the mature protein (amino acid residues 21- 105 of SEQ ID NO: 19) is encoded by nucleic acid residues 88-342 of SEQ ID NO: 20.

The LJM06 unprocessed protein (SEQ ID NO: 21) is encoded by nucleic acid residues 50-523 of SEQ ID NO: 22, and the mature protein (amino acid residues 20- 157of SEQ ID NO: 21) is encoded by nucleic acid residues 107-523 of SEQ ID NO: 22.

The LJM17 unprocessed protein (SEQ ID NO: 23) is encoded by nucleic acid residues 24-1264 of SEQ ID NO: 24, and the mature protein (amino acid residues 19-412 of SEQ IDNO: 23) is encoded by nucleic acid residues 83-1264 of SEQ ID NO: 24.

The LJL04 unprocessed protein (SEQ ID NO: 25) is encoded by nucleic acid residues 30-914 of SEQ ID NO: 26, and the mature protein (amino acid residues 18- 295 of SEQ ID NO: 25) is encoded by nucleic acid residues 81-914 of SEQ ID NO: 26.

The LJMl 14 unprocessed protein (SEQ ID NO: 27) is encoded by nucleic acid residues 29-475 of SEQ ID NO: 28, and the mature protein (amino acid residues 25-148 of SEQ ID NO: 27) is encoded by nucleic acid residues 101-475 of SEQ ID NO: 28.

The LJMlI l unprocessed protein (SEQ ID NO: 29) is encoded by nucleic acid residues 24-1214 of SEQ ID NO: 30, and the mature protein (amino acid residues 19-397 of SEQ ID NO: 29) is encoded by nucleic acid residues 78-1214 of SEQ ID NO: 30.

The LJM78 mature unprocessed protein (SEQ ID NO: 31) is encoded by nucleic acid residues 42-1091 of SEQ ID NO: 32, and the mature protein (amino acid residues 21-350 of SEQ ID NO: 31) is encoded by nucleic acid residues 102- 11091 of SEQ ID NO: 32.

The LJS238 unprocessed protein (SEQ ID NO: 33) is encoded by nucleic acid residues 27-206 of SEQ ID NO: 34, and the mature protein (amino acid residues 21-60 of SEQ ID NO: 33) is encoded by nucleic acid residues 87-206 of SEQ ID NO: 34.

The LJS169 unprocessed protein (SEQ ID NO: 35) is encoded by nucleic acid residues 11-370 of SEQ ID NO: 36, and the mature protein (amino acid residues 23-120 of SEQ ID NO: 35) is encoded by nucleic acid residues 77-370 of SEQ ID NO: 36.

The LJLIl unprocessed protein (SEQ ID NO: 37) is encoded by nucleic acid residues 30-1745 of SEQ ID NO: 38, and the mature protein (amino acid residues 26-572 of SEQ ID NO: 37) is encoded by nucleic acid residues 105-1745 of SEQ ID NO: 38.

The LJL08 unprocessed protein (SEQ ID NO: 39) is encoded by nucleic acid residues 26-238 of SEQ ID NO: 40, and the mature protein (amino acid residues 24- 86 of SEQ ID NO: 39) is encoded by nucleic acid residues 95-238 of SEQ ID NO: 40. The LJS 105 unprocessed protein (SEQ ID NO: 41) is encoded by nucleic acid residues 24-275 of SEQ ID NO: 42, and the mature protein (amino acid residues 20-84 of SEQ ID NO: 41) is encoded by nucleic acid residues 81-275 of SEQ ID NO: 42.

The LJL09 unprocessed protein (SEQ ID NO: 43) is encoded by nucleic acid residues 74-1954 of SEQ ID NO: 44, and the mature protein (amino acid residues 19-626 of SEQ ID NO: 43) is encoded by nucleic acid residues 128-1954 of SEQ ID NO: 44.

The LJL38 unprocessed protein (SEQ ID NO: 45) is encoded by nucleic acid residues 40-165 of SEQ ID NO: 46, and the mature protein (amino acid residues 21- 42 of SEQ ID NO: 45) is encoded by nucleic acid residues 100-165 of SEQ ID NO: 46.

The LJM04 unprocessed protein (SEQ ID NO: 47) is encoded by nucleic acid residues 40-456 of SEQ ID NO: 48, and the mature protein (amino acid residues 21- 139 of SEQ ID NO: 47) is encoded by nucleic acid residues 100-456 of SEQ ID NO: 48.

The LJM26 unprocessed protein (SEQ ID NO: 49) is encoded by nucleic acid residues 96-1616 of SEQ ID NO: 50, and the mature protein (amino acid residues 18-446 of SEQ ID NO: 49) is encoded by nucleic acid residues 147-1616 of SEQ ID NO: 50.

The LJS03 unprocessed protein (SEQ ID NO: 51) is encoded by nucleic acid residues 41-553 of SEQ ID NO: 52, and the mature protein (amino acid residues 20- 166 of SEQ ID NO: 51) is encoded by nucleic acid residues 98-553 of SEQ ID NO: 52.

The LJS192 unprocessed protein (SEQ ID NO: 53) is encoded by nucleic acid residues 18-344 of SEQ ID NO: 54, and the mature protein (amino acid residues 24-109 of SEQ ID NO: 53) is encoded by nucleic acid residues 87-344 of SEQ ID NO: 54.

The LJM19 unprocessed protein (SEQ ID NO: 55) is encoded by nucleic acid residues 16-360 of SEQ ID NO: 56, and the mature protein (amino acid residues 23- 115 of SEQ ID NO: 55) is encoded by nucleic acid residues 82-360 of SEQ ID NO: 56.

The LJLl 38 unprocessed protein (SEQ ID NO: 57) is encoded by nucleic acid residues 12-1238 of SEQ ID NO: 58 and the mature protein (amino acid residues 21-409 of SEQ ID NO: 57) is encoded by nucleic acid residues 72-1238 of SEQ ID NO: 58.

The LJL15 unprocessed protein (SEQ ID NO: 59) is encoded by nucleic acid residues 63-542 of SEQ ID NO: 60, and the mature protein (amino acid residues 20- 160 of SEQ ID NO: 59) is encoded by nucleic acid residues 120-542 of SEQ ID NO: 60.

The LJL91 unprocessed protein (SEQ ID NO: 61) is encoded by nucleic acid residues 63-542 of SEQ ID NO: 62, and the mature protein (amino acid residues 20- 160 of SEQ ID NO: 61) is encoded by nucleic acid residues 120-542 of SEQ ID NO: 62).

The LJMIl unprocessed protein (SEQ ID NO: 63) is encoded by nucleic acid residues 20-1216 of SEQ ID NO: 64, and the mature protein (amino acid residues 19-399 of SEQ ID NO: 63) is encoded by nucleic acid residues 74-1216 of SEQ ID NO: 64.

The LJS138 unprocessed protein (SEQ ID NO: 65) is encoded by nucleic acid residues 12-1238 of SEQ ID NO: 66, and the mature protein (amino acid residues 21-170 of SEQ ID NO: 65) is encoded by nucleic acid residues 72-138 of SEQ ID NO: 66.

The LJL 124 unprocessed protein (SEQ ID NO: 67) is encoded by nucleic acid residues 23-241 of SEQ ID NO: 68, and the mature protein (amino acid residues 21-73 of SEQ ID NO: 67) is encoded by nucleic acid residues 83-241 of SEQ ID NO: 68.

The LJL35 unprocessed protein (SEQ ID NO: 69) is encoded by nucleic acid residues 12-1238 of SEQ ID NO: 70, and the mature protein (amino acid residues 24-76 of SEQ ID NO: 69) is encoded by nucleic acid residues 72-1238 of SEQ ID NO: 70.

The above identified Lu. longipalpis polypeptides are further characterized in Valenzuela et al, J. Exp. Bio., 207:3717-3729, 2004, which is incorporated herein by reference.

Antibodies

An anti-complement polypeptide of the disclosure or a fragment thereof according to the disclosure can be used to produce antibodies. Polyclonal antibodies, antibodies which consist essentially of pooled monoclonal antibodies with different epi topic specificities, as well as distinct monoclonal antibodies are included.

The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al , "Production of Polyclonal Antisera," Immunochemical Protocols, pp. 1-5, Manson, ed., Humana Press, 1992; Coligan et al, "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters," Current Protocols in Immunology, section 2.4.1, 1992.

The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature 256:495, 1975; Coligan et al, sections 2.5.1- 2.6.7; and Harlow et al, Antibodies: A Laboratory Manual, p. 726, Cold Spring Harbor Pub., 1988. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well- established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan et al , sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al, "Purification of Immunoglobulin G (IgG)," Methods in Molecular Biology, Vol. 10, pp. 79-104, Humana Press, 1992.

Methods of in vitro and in vivo multiplication of monoclonal antibodies are well known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally supplemented by a mammalian serum such as fetal calf serum or trace elements and growth- sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes, or bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large-scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, for example, syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

Antibodies can also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in WO 91/11465, 1991, and Losman et al, Int. J. Cancer 46:310, 1990.

Alternatively, an antibody that specifically binds a polypeptide can be derived from a humanized monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al. , Proc. Nat'l Acad. ScL USA 86:3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. ScL USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al. , J. Immunol. 150:2844, 1993.

Antibodies can be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al. , Methods: a Companion to Methods in Enzytnology, Vol. 2, p. 119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).

In addition, antibodies can be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et ah, Nature Genet. 7:13, 1994; Lonberg et al , Nature 368:856, 1994; and Taylor et al, Int. Immunol. 6:579, 1994.

Antibodies include intact molecules as well as fragments thereof, such as Fab, F(ab')₂, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with their antigen or receptor and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain (L) and a portion of one heavy chain(H);

(2) Fab', the fragment of an antibody molecule that can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')₂, the fragment of the antibody that can be obtained by treating a whole antibody with the enzyme pepsin without subsequent reduction; F(ab')₂ is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain (V_L) and the variable region of the heavy chain (V_H) expressed as two chains; and

(5) Single chain antibody (SCA), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).

Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')₂. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al. , Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73: 119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

For example, Fv fragments comprise an association of V_H and V_L chains. This association may be noncovalent (Inbar et al. , Proc. Nat 'I Acad. ScL USA 69:2659, 1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, for example, Sandhu, supra. In one embodiment, the Fv fragments comprise V_H and V_L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V_H and V_L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are known in the art (see Whitlow et al. , Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al, Bio/Technology 11: 1271, 1993; and Sandhu, supra).

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (Larrick et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106, 1991).

Antibodies can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or a peptide used to immunize an animal can be derived from substantially purified polypeptide produced in host cells, in vitro translated cDNA, or chemical synthesis which can be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize an animal (for example, a mouse, a rat, or a rabbit).

Polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991).

It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the "image" of the epitope bound by the first monoclonal antibody.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, label (for example, enzymes or fluorescent molecules) drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

In one embodiment, an antibody that binds an anti-complement polypeptide can be used to assess whether a subject has been bitten by a sand fly. In one specific, non-limiting example, a sample is obtained from a subject of interest, such as a human or a dog. The sample can be a body fluid (for example, blood, serum, urine, saliva, etc.) or a tissue biopsy. The sample or a fraction thereof is contacted with the antibody, and the ability of the antibody to form an antigen-antibody complex is assessed. One of skill in the art can readily detect the formation of an antigen- antibody complex. For example, ELISA, Western blot, or radio-immune assays can be utilized.

Methods of Use of Anti- Complement Compositions

The anti-complement polypeptides disclosed herein, and the nucleic acid molecules encoding the anti-complement polypeptides, can be used to modulate polypeptides of the complement system either in vitro or in vivo. For example, the activity of a complement polypeptide can be increased or decreased as a result of interaction or contact with an anti-complement composition disclosed herein. The complement polypeptide can be a classical pathway polypeptide, an alternative pathway polypeptide, a lectin pathway polypeptide, or a polypeptide common to a combination of the pathways. In specific embodiments, the disclosed anti- complement composition has an inhibitory effect (for example, a specific inhibitory effect) on the complement system by inhibiting or reducing the activity of a complement component, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5. In particular embodiments, anti-complement compositions disclosed herein can be used to prevent and treat diseases and pathologies associated with increased, undesirable, or abnormal complement activation. In addition, anti-complement polypeptides can be used to generate a transmission-blocking vaccine, which would block the portion of the leishmania life cycle that takes place in the sand fly, thereby preventing the infection of an organism, such as a human or a dog. Thus, the anti- complement compositions disclosed herein have a wide range of medical applications, in the treatment, prevention and diagnosis of diseases and conditions, as well as being useful in the study of complement inhibition and of the inhibition of the alternative, lectin, and classical pathways of complement activation.

Use of Anti-Complement Compositions In Vitro

Provided herein are methods for inhibiting (for example, specifically inhibiting) the classical, alternative, or lectin complement pathways in an in vitro sample by contacting the sample with an anti-complement polypeptide, a fusion protein comprising an anti-complement polypeptide, or a nucleic acid molecule encoding the anti-complement polypeptide, as disclosed herein. The sample can be from a normal (control) subject, for example a subject who is not suffering from a disease or a pathology associated with increased, undesirable, or abnormal activation of a component of the complement system, or from a subject who is suffering from a disease or a pathology associated with increased, undesirable, or abnormal activation of a component of the complement system. The sample can be a blood sample, a plasma sample, a cell sample, or a tissue sample.

The ability of a molecule to modulate the activity of a component of the complement system can be determined by standard hemolytic assays known in the art, such as those described in the Examples and in Giclas et al. (Classical and alternative pathway evaluation (sections 13.1 and 13.2). In Current Protocols in Immunology, Vol. 3, Complement. Editors: J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M.1994). In one embodiment, the presence of a disclosed anti- complement composition reduces red blood cell lysis in standard hemolytic assays of complement activation by at least 20%, compared to a standard assay in the absence of an anti-complement composition. In other embodiments, the presence of a disclosed anti-complement composition reduces red blood cell lysis in standard hemolytic assays of complement activation by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, compared to a standard assay in the absence of an anti- complement composition. In a specific non-limiting example, a reduction in cell lysis correlates with an anti-complement composition modulating the activity of a complement molecule, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5. For example, the activity of a complement molecule can be decreased or increased as a result of interaction with the salivary gland polypeptides disclosed herein. In one specific, non- limiting example, the inhibition of C3b activity inhibits the formation of C5 convertase and prevents the conversion of C5 complement component into C5a and C5b. Thus, the disclosed methods can be used as a diagnostic tool to identify molecules that modulate the activity of components of the complement system

In Vivo Administration to Treat Disease in Subject

Abnormalities in any one of the many components of the complement cascade can result in an altered immune response in a subject. Particular diseases and pathologies are believed to have a complement-mediated component (such as unwanted activation of the complement pathway, such as the classical complement pathway. These diseases include septic shock, complement activation during cardiopulmonary bypass surgery (due, for example, to interaction of blood with the extracorporeal circuit of the heart-lung machine used during such surgeries), systemic lupus erythematosus (SLE) (lupus nephritis and resultant glomerulonephritis and vasculitis), rheumatoid arthritis (RA), juvenile chronic arthritis, adult respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, pemphigus, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulemia, antiphospholipid syndrome, age-related macular degeneration, uveitis, diabetic retinopathy, allotransplantation, hemodialysis, chronic occlusive pulmonary disetress syndrome (COPD), asthma, and aspiration pneumonia, inflammatory bowel disease (IBD), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), transplantation associated diseases including graft rejection and graft- versus host disease as well as other pathologies and diseases (see Markiewski et ah, Am. J. Path, 171:715-727, 2007, incorporated by reference).

In particular pathologies, such as glomerulonephritis, complement is deposited in the kidney, for example (1) within the glomerular mesangium, as in IgA nephropathy, Henoch-Schonlein purpura, and early lupus nephritis; (2) along the subendothelial surface of the capillary wall between endothelial cells and glomerular basement membrane, as seen in more severe forms of lupus nephritis and type I membranoproliferative glomerulonephritis; and (3) on the outer, subepithelial surface of the capillary wall, as in membranous nephropathy and poststreptococcal glomerulonephritis. Thus, it is of interest to develop inhibitors to control the complement system, and to prevent and treat diseases and pathologies, such as those described above.

In accordance with the present disclosure, the anti-complement compositions provided herein are useful for inhibiting or reducing complement activation (for example, inhibiting or reducing the activity of a component of the complement system, such as a specific inhibitor of the classical complement pathway) in a subject. In particular embodiments, the sand fly salivary gland polypeptides disclosed herein (for example a Lu. longipalpis polypeptide, such as residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63. or variants or fragments thereof) specifically bind a component of the complement system (such as, but not limited to, C2, C4, C4b, C2a, C3, C3b, or C5) and inhibit or reduce activity of the component, thereby inhibiting or reducing complement activation and complement mediated effector functions that, when activated, can lead to tissue damage and disease in the subject. The administration to a subject of a disclosed anti-complement composition inhibits or reduces complement activity in a subject. In particular embodiments, a subject with excess, undesirable, or abnormal complement activation is selected for treatment. In other embodiments, the sand fly salivary gland anti-complement composition is not administered to a subject with or for the purpose of treating a leishmania infection in the subject.

When administered to a subject, an anti-complement composition disclosed herein inhibits or reduces the activity of a component of the complement system, thereby inhibiting or reducing the activity of the complement system in a subject. Thus, anti-complement compositions can be used for the prevention or treatment of a disease or pathology resulting from an immune response caused by complement activation, thereby preventing or treating the disease or pathology. In one embodiment, the disclosed anti-complement polypeptides and nucleic acid sequences encoding the polypeptides can be used for the treatment or prevention of disorders characterized by the abnormal or undesirable activation of one or more components of the classical, alternative, or lectin pathways. The administration to a subject of a disclosed anti-complement composition decreases activity of the complement component and thereby decreases the severity and/or length of time of the immune response related to the activation of the complement system in the subject.

An inhibition (or reduction) of complement activity can be measured by any standard method known to one of skill in the art. In one embodiment, a decrease in cell lysis (as measured by a hemolytic assay) when a cell sample is contacted with a sample obtained from a subject and a sand fly salivary gland polypeptide, compared to a cell sample in contact with a sample obtained from the subject in the absence of a sand fly salivary gland polypeptide, is an indication that the polypeptide has anti- complement activity. The sample obtained from the subject can be from a normal (control) subject, for example a subject who is not suffering from a disease or a pathology associated with increased, undesirable, or abnormal activation of a component of the complement system, or from a subject who is suffering from a disease or a pathology associated with increased, undesirable, or abnormal activation of a component of the complement system. In some embodiments, the reduction or inhibition of complement activity is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In yet other embodiments, an inhibition or reduction in complement activity is measured by a clinical response in vivo, such as a measurable reduction in symptoms related to various pathologies or syndromes, such as asthma, lupus erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, and ischemia-reperfusion injuries.

Generation of Transmission-Blocking Antibodies

Anti-complement polypeptides found in the saliva of sand flies can be re- ingested by the sand fly during a blood meal, thereby protecting the sand fly midgut from the damaging effects of complement proteins present in the blood meal. When the sand fly takes a blood meal from a host, the ingested blood is surrounded by a peritrophic matrix within 12-24 hours. The matrix consists of various proteins and chitin fibrils to form a lattice-work around the blood bolus. The biological importance of the peritrophic matrix is to help protect the luminal surface of the midgut from food particles and to compartmentalize digestion. When a sand fly acquires a blood meal infected with parasites, the parasites are within the blood bolus, surrounded by the peritrophic matrix. Thus, the matrix protects the early stage parasites from being damaged by the sand fly's digestive proteases. However, leishmania parasites, which are sensitive to the action of human and dog complement, may be additionally protected in the sand fly midgut by sand fly anti- complement polypeptides, for example Lu. longipalpis salivary gland polypeptides, such as residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or variants or fragments thereof. Thus, inhibiting the activity of the anti-complement polypeptides in the midgut of a sand fly would be lethal to the parasite and block further transmission of the parasite.

It is therefore provided herein that the sand fly salivary gland anti- complement polypeptides disclosed herein (or the polynucleotides encoding these polypeptides) can be used to generate inhibitory antibodies directed against the polypeptides (antibodies that block the activity of the anti-complement polypeptides). A multitude of techniques are available to those skilled in the art for production and manipulation of various immunoglobulin molecules (as discussed above) that can be readily used to block transmission of a parasite.

In one specific embodiment, the sand fly salivary gland anti-complement polypeptides disclosed herein (or the polynucleotides encoding these polypeptides) can be used to generate a transmission blocking immune response in an organism, for example a human or a dog. The organism can be currently infected by leishmania (either symptomatic or asymptomatic) or at risk of being infected by leishmania. In one particular, non-limiting embodiment, a subject is administered an immunologically effective amount of a polypeptide having at least 90% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55 in order to induce an immune response against residues 23-115 of SEQ ID NO: 55 in the subject. Antibodies produced in the subject specifically bind the polypeptide set forth as residues 23-115 of SEQ ID NO: 55. When ingested by a sand fly during a blood meal, the antibodies bind the polypeptide set forth as residues 23-115 of SEQ ID NO: 55 and inhibit its anti-complement activity, thus exposing leishmania parasites in the sand fly midgut to the damaging effects of complement proteins present there. Leishmania parasites are therefore prevented from developing and completing their life cycle in the sand fly midgut and further transmission of the parasite is blocked. A reduction in parasite load in a sand fly which has fed on a subject immunized with the polypeptide set forth as residues 23-115 of SEQ ID NO: 55, compared to the parasite load in a sand fly which has fed on a subject that has not been immunized with the polypeptide, is a measure of the effectiveness of a transmission blocking vaccine. A reduction in parasite load can be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. Pharmaceutical and Immunogenic Compositions

The disclosed polypeptides can be formulated in pharmaceutical or immunogenic compositions. In one embodiment, the anti-complement composition includes a therapeutically or immunologically effective amount of at least one sand fly salivary gland polypeptide disclosed herein. In one specific embodiment, the anti-complement composition includes a sand fly salivary gland polypeptide, for example a Lu. longipalpis salivary gland polypeptide having an amino acid sequence as set forth as residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or variants or fragments thereof. In other specific embodiments, the anti-complement composition includes a polypeptide having an amino acid sequence as set forth as SEQ ID NO: 47, SEQ ID NO; 49, SEQ ID NO: 55, or SEQ ID NO: 63, or variants or fragments thereof. In yet other embodiments, the anti-complement composition includes a sand fly salivary gland polypeptide, having at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% homology to the polypeptides disclosed herein, a conservative variant or a homolog thereof, or a fragment thereof, such as a fragment comprising at least eight, at least nine, at least ten, at least eleven, or at least twelve consecutive amino acids of one of these polypeptides, or a combination of these polypeptides.

In another embodiment, the anti-complement composition includes an effective amount of one or more sand fly salivary gland polypeptides. Examples of these polypeptide sequences are disclosed in PCT/US2003/034453 filed October 29, 2003; PCT Application No. PCT/US2003/029833, filed September 18, 2003; and PCT/US02/19663, filed June 18, 2002, which are incorporated herein by reference.

The polynucleotides disclosed herein (for example, a polynucleotide encoding a Lu. longipalpis polypeptide, or a polynucleotide encoding residues 21- 139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63) also can be formulated in pharmaceutical compositions. In other embodiments, the polynucleotide encoding an anti-complement polypeptide is a polynucleotide having at least 75%, 85%, 90%, 95%, or 99% homology to a nucleotide sequence that encodes an anticoagulant polypeptide. In one embodiment, the anti-complement composition comprises an effective amount of a recombinant vector expressing at least one sand fly salivary gland polypeptide disclosed herein. The anti-complement composition can include a nucleic acid sequence encoding two or more sand fly salivary gland polypeptides. In one embodiment, the two or more sand fly salivary gland polypeptides are encoded by the same recombinant vector. In another embodiment, the two or more polypeptides are encoded by different recombinant vectors.

The pharmaceutical and immunogenic compositions may comprise, in addition to one of the above substances, a pharmaceutically or immunologically acceptable vehicle, excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal.

The anti-complement polypeptides, and polynucleotides encoding these anti- complement polypeptides, can be administered by any means known to one of skill in the art (See Banga, A., "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995) such as by intramuscular (IM), intradermal (ID), subcutaneous (SC), or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous, intradermal, or intramuscular injection using a needleless injector (Biojector™, Bioject, Oregon, USA).

To extend the time during which the sand fly salivary gland polypeptide is available to inhibit complement activation, the peptide or protein can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle. (see, for example, Banja, supra). A particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release. An anti-complement composition according to the disclosure can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary art. Administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. A therapeutically effective amount of an anti-complement composition is an amount used to inhibit or reduce complement activity. In another embodiment, a therapeutically effective amount of an anti-complement composition is an amount used to inhibit or reduce the activity of a component of the complement system, such as, but not limited to, C2, C4, C4b, C2a, C3, C3b, or C5. In yet another embodiment, a therapeutically effective amount of an anti-complement composition is an amount sufficient to prevent or treat a disorder associated with increased, undesirable, or abnormal complement activity in a subject.

Vaccine compositions containing the sand fly salivary gland polypeptides or polynucleotides disclosed herein are administered to a subject to elicit a transmission-blocking immune response against the polypeptide (antigen) and thus prevent spread of the disease through the sand fly vector. Such an amount is defined as an immunogenically effective dose. 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 therapeutically effective fragment of an anti-complement polypeptide includes any fragment of the sand fly salivary gland polypeptide, or variant of the sand fly salivary gland polypeptide, or fusion protein including a sand fly salivary gland polypeptide, that retains a function of the sand fly salivary gland polypeptide (such as inhibiting complement activity), or retains the ability to reduce the symptoms related to a disorder associated with increase, undesirable, or abnormal complement activity. Thus, in one embodiment, a therapeutically effective amount of an anti-complement polypeptide or a fragment of an anti-complement polypeptide is an amount used to bind to a component of the complement system, or to inhibit or reduce the activity of such a component. In another embodiment, a therapeutically effective amount of a fragment of sand fly salivary gland polypeptide is an amount of use to prevent or decrease conversion of C3 into C3a and C3b, and inhibiting or reducing complement activity. In yet another embodiment, a therapeutically effective amount of a fragment of a sand fly salivary gland polypeptide is an amount to prevent or treat a disorder associated with an increased, undesirable, or abnormal complement activity in a subject. An immunologically effective fragment of an anti- complement polypeptide includes any fragment of the sand fly salivary gland polypeptide, or variant of the sand fly salivary gland polypeptide, or fusion protein including a sand fly salivary gland polypeptide, that retains the immunogenic epitope of the sand fly salivary gland polypeptide. Specific, non-limiting examples of a polypeptide fragment are the N-terminal half or the C-terminal half of one of the sand fly salivary gland polypeptide disclosed herein. In one embodiment, a therapeutically effective or immunologically effective fragment of an anti- complement polypeptide includes at least 8, 10, 15, 19, 20 23, 25, or 30 amino acids of an anti-complement polypeptide. It should be noted that fusion proteins are included, such as a fusion with six histidine residues, a c-myc tag, or any other polypeptide tag. Such fusions are known to one of skill in the art, and are often used in protein purification.

A sample obtained from a subject who is administered a therapeutically effective amount of an anti-complement composition can be tested for change in cell lysis, compared to a sample from a subject that has not been administered the anti- comlement composition. A reduction in cell lysis can be at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100%, compared to a control sample. Similarly, inhibiting complement activity of a sample obtained from a subject who is administered a therapeutically effective amount of an anti- complement composition can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% inhibition, compared to a subject who has not been administered a therapeutically effective amount of the anti-complement composition.

The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species, and condition of the particular subject, and the route of administration. The route of administration can be via any route that delivers a safe and therapeutically effective dose of a composition of the present disclosure to the animal or human. Forms of administration, include, but are not limited to, topical, enteral, and parenteral routes of administration. Enteral routes include oral and gastrointestinal administration. Parenteral routes include intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, transdermal, and transmucosal administration. Other routes of administration include epidural or intrathecal administration. The effective dosage and route of administration are determined by the therapeutic range and nature of the compound, and by known factors, such as the age, weight, and condition of the patient, as well as LD50 and other screening procedures that are known and do not require undue experimentation. Examples of the techniques and protocols mentioned above can be found in Remington 's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The term "dosage" as used herein refers to the amount of an anti-complement composition administered to an animal or human. The therapeutic agent may be delivered to the recipient as a bolus or by a sustained (continuous or intermittent) delivery. When the delivery of a dosage is sustained over a period, which may be in the order of a few minutes to several days, weeks or months, or may be administered chronically for a period of years, the dosage may be expressed as weight of the therapeutic agent/kg body weight of the subject/unit time of delivery.

In one embodiment of the present disclosure, an anti-complement composition is administered as a bolus to a subject in need thereof, to inhibit or reduce complement activation, in a dose of about 0.1 ng to about 500 mg per kg of body weight, about 10 ng to about 300 mg per kg of body weight, from about 100 ng to about 200 mg per kg of body weight, from about 1 μg to about 100 mg per kg of body weight, from about 1 μg to about 50 mg per kg of body weight, or from about 1 μg to about 1 mg per kg of body weight. Alternatively, the amount of an anti- complement composition administered to achieve a therapeutically effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, or 50 mg per kg of body weight or greater. In specific, non- limiting examples, the anti- complement composition is residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or a variant thereof, or is residues 100-456 of SEQ ID NO: 48, residues 147- 1616 of SEQ ID NO; 50, residues 82-360 of SEQ ID NO: 56, or residues 74-1216 of SEQ ID NO: 64, or a degenerate variant thereof, and is administered parenterally, preferably intravenously.

In another embodiment, an antic-complement composition is administered continuously to a subject in need thereof, to inhibit or reduce complement activation, in a dose of about 0.1 ng, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, or 1 mg per kg of body weight per minute or greater. In specific, non-limiting examples, the anti-complement composition is residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or a variant thereof, or is residues 100-456 of SEQ ID NO: 48, residues 147-1616 of SEQ ID NO; 50, residues 82-360 of SEQ ID NO: 56, or residues 74-1216 of SEQ ID NO: 64, or a degenerate variant thereof, and is administered parenterally, preferably intravenously.

In yet another embodiment, an anti-complement compoistion is administered to a patient in need thereof, to inhibit or reduce complement activation, in a dose sufficient to achieve a blood plasma concentration of 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 11 μg/ml, 12 μg/ml, 13 μg/ml, 14 μg/ml, 15 μg/ml, 16 μg/ml, 17 μg/ml, 18 μg/ml, 19 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg/ml, 90 μg/ml, or 100 μg/ml or greater. In specific, non- limiting examples, the anti-complement composition is residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or a variant thereof, or is residues 100-456 of SEQ ID NO: 48, residues 147-1616 of SEQ ID NO; 50, residues 82-360 of SEQ ID NO: 56, or residues 74-1216 of SEQ ID NO: 64, or a degenerate variant thereof, and is administered parenterally, preferably intravenously.

In several embodiments, polypeptide dosages can be from about 1 to 250 μg/ml, from about 15 to about 150 μg/dose, or from about 20 to about 100 μg/dose. In another embodiment, using a needle-less apparatus the volume of a dose can be between about 0.1 ml and about 0.5 ml. In yet another embodiment, using a needleless apparatus the volume of a dose can be about 0.25 ml. Administration with multiple points of injection is preferred. In one embodiment, for conventional injection with a syringe and a needle, the volumes are from about 0.1 to about 2 ml. In another embodiment, for conventional injection with a syringe and a needle, the volumes are from about 0.5 to about 1 ml.

For plasmid-based compositions, the route of administration can be ID, IM, SC, intravenous, oral, nasal, or anal. This administration can be made with a syringe and a needle or with a needle-less apparatus like, for example, Biojector™. The dosage is from about 50 μg to about 500 μg per plasmid. When DMRIE-DOPE is added, about 100 μg per plasmid is preferred. In one embodiment, when canine GM-CSF or other cytokine is used, the plasmid encoding this protein is present at a dosage from about 200 μg to about 500 μg. In another embodiment, the plasmid encoding this protein is present at a dosage of about 200 μg. In one embodiment, using a needle-less apparatus, the volume of a dose can be between about 0.1 ml and about 0.5 ml. In another embodiment, the volume of a dose can be about 0.25 ml. In yet another embodiment, administration is performed using multiple points of injection. In one embodiment, for conventional injection with a syringe and a needle, the volumes are from about 0.1 to about 2 ml. In another embodiment, the volumes are from about 0.5 to about 1 ml. The dosages are the same as those mentioned above.

For recombinant viral vector-based compositions, the route of administration can be ID, IM, SC, intravenous, oral, nasal, or anal. This administration can be made with a syringe and a needle or with a needle-less apparatus like, for example, Biojector™. The dosage is from about 10³ pfu to about 10⁹ pfu per recombinant poxvirus vector. In one embodiment, when the vector is a canarypox virus, the dosage is from about 10⁵ pfu to about 10⁹ pfu. In another embodiment, the dosage is from about 10⁶ pfu to about 10⁸ pfu. In one embodiment, the volume of needle-less apparatus doses could be between about 0.1 ml and about 0.5 ml. In another embodiment, the volume of needle-less apparatus dose is 0.25 ml. In yet another embodiment, administration is performed using multiple points of injection. In one embodiment, for conventional injection with a syringe and a needle, the volumes are from about 0.1 to about 2 ml. In another embodiment, the volumes are from about 0.5 to about 1 ml. The dosages are the same as mentioned above. In one embodiment, when a syringe with a needle is used, the injection is EVI.

A typical treatment course can comprise about six doses delivered over a 7 to 21 day period. Upon election by the clinician, the regimen can be continued six doses every three weeks or on a more frequent (daily, twice daily, four times a day, etc.) or less frequent (monthly, bimonthly, quarterly, etc.) basis. Of course, these are only exemplary times for treatment, and the skilled practitioner will readily recognize that many other time-courses are possible. The anti-complement compositions can be combined with any of a number of conventional treatment regimens. Regional delivery of sand fly salivary gland anti-complement compositions is an efficient method for delivering a therapeutically effective dose to counteract the clinical disease.

Compositions comprising the disclosed sand fly salivary polypeptide or a polynucleotide encoding the sand fly salivary polypeptide (anti-complement composition) may be administered alone or in combination with other anti- complement treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of other anti-complement agents include leupeptin and eculizumab (a monoclonal antibody directed against the complement protein C5). Combination therapies are provided in which an anti-complement composition disclosed herein is the primary active agent and is administered along with an additional active agent to a subject in order to inhibit or reduce complement activity in the subject. Such combination therapy may be carried out by administration of the different active agents in a single composition, by concurrent administration of the different active agents in different compositions, or by sequential administration of the different active agents. The additional active agent will generally, although not necessarily, be one that enhances or potentiates the effect of the salivary gland anti- complement polypeptide.

Methods of Immunization

The present disclosure provides methods for inducing an immune response to a sand fly polypeptide in a subject, for example to generate a transmission blocking vaccine against leishmania. These methods include the administration of at least one immunogenic composition or vaccine according to the disclosure.

If more than one administration is required, they can be administered concurrently (for example, different compositions given during the same period of time via the same or different routes, or a same composition given in the same period of time via different routes), or sequentially (for example, the same or different compositions given at least two times via the same or different routes). In one embodiment, the delay between two sequential administrations is from about 1 week to about 6 months. In another embodiment, the delay is from about 3 weeks to about 6 weeks. In yet another embodiment, the delay is from about 4 weeks. Following vaccination, annual boost administrations may be done. Advantageously, in a prime-boost vaccination schedule, at least one prime-administration can be done with a composition containing a plasmid according to the disclosure, following by at least one booster administration done with a composition containing a recombinant viral vector according to the disclosure, on the condition that a same sand fly salivary gland polypeptide is present twice, coded by the plasmid and by the viral vector. Alternatively, the booster administration can be done with a composition containing a polypeptide according to the disclosure, on the condition that a same sand fly salivary gland polypeptide is present twice, coded by the prime- administration plasmid and in the booster polypeptide-based composition. In such compositions the antigen(s) may be in admixture with a suitable vehicle or excipient such as sterile water, physiological saline, glucose, or the like. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling, or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as Remington's Pharmaceutical Science, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. The compositions can also be lyophilized.

Advantageously for the prime boost administration regimen, the prime- administration is made with a plasmid-based composition and the boost administration is made with a recombinant viral vector-based composition. In one embodiment, the boost administration is made with a canarypox vector. Both priming and boosting administrations include vectors encoding at least one identical sand fly salivary gland antigen and optionally Leishmania A2 antigens. The dosage of plasmids and recombinant viral vectors are the same as above. Optionally, the boost administration can be done with a polypeptide-based composition. In this case, the dosage of polypeptide is from about 1 to about 250 μg/ml, from about 15 to about 150 μg/dose, or from about 20 to about 100 μg/dose.

Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response) and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune- stimulating constructs, or ISCOMS ™, negatively charged cage-like structures of 30- 40 nm in size formed spontaneously on mixing cholesterol and Quil A™ (saponin). Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS ™ as the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS ™ have been found to produce class I mediated CTL responses (Takahashi et al. , Nature 344:873, 1990).

In another approach using nucleic acids for immunization, a sand fly salivary gland polypeptide, or an immunogenic fragment thereof, can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, or other viral vectors can be used to express the peptide or protein, thereby eliciting a CTL response. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a sand fly salivary gland polypeptide, or an immunogenic fragment thereof, is introduced directly into cells. For example, the nucleic acid may be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad's Helios™ Gene Gun. A needless injector can also be utilized, such as a Bioinjector2000™. The nucleic acids can be "naked," consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Exemplary dosages for injection are around 0.5 μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, for example, U.S. Patent No. 5,589,466). In one embodiment, a prime-boost strategy for immunization is utilized. Thus, in one embodiment, a nucleic acid encoding a sand fly salivary gland polypeptide is administered to the subject, followed by immunization with an attenuated or inactivated form of leishmania.

The immunogenic compositions and the vaccines disclosed herein can be administered for preventative and therapeutic treatments. In therapeutic applications, compositions are administered to a subject suffering from a disease, such as leishmaniasis, in a therapeutically effective amount, which is an amount sufficient to cure or at least partially arrest the disease or a sign or symptom of the disease. Amounts effective for this use will depend upon the severity of the disease and the general state of the subject's health. An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.

Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject.

As noted above, the dosage of the composition varies depending on the weight, age, sex, and method of administration. The dosage can also be adjusted by the individual physician as called for based on the particular circumstances. The compositions can be administered conventionally as vaccines containing the active composition as a predetermined quantity of active material calculated to produce the desired therapeutic or immunologic effect in association with the required pharmaceutically acceptable carrier or diluent (for instance, carrier or vehicle). For example, about 50 μg of a DNA construct vaccine of the present disclosure can be injected intradermally three times at two week intervals to produce the desired therapeutic or immunologic effect. In another embodiment, a about 1 mg/kg dosage of a protein vaccine of the present disclosure can be injected intradermally three times at two week intervals to produce the desired therapeutic or immunologic effect.

A vaccine is provided herein that includes a sand fly salivary gland polypeptide or polynucleotide. Administration of the vaccine to a subject, such as a human or veterinary subject, results in an immune response to a sand fly salivary gland anti-complement polypeptide, for example to generate a transmission blocking vaccine against leishmania. In one embodiment, the subject is a human subject. In another embodiment, the subject is a canine subject, such as a dog.

Method of Screening for an Agent that Inhibits Complement Activation

Methods are provided for screening agents that inhibit complement activation. Thus, methods are disclosed herein for identifying anti-complement polypeptides that have an inhibitory effect on a component of the complement system, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5.

In one specific embodiment, a sample (for example a plasma sample) is contacted with an agent of interest (for example, a sand fly salivary gland polypeptide disclosed herein), and the effect of the agent on the complement activity of the sample (for example, percent blood cell lysis) is then assayed and compared to a control sample that has not been contacted with the agent. A decrease in cell lysis indicates that the agent inhibits a component of the complement system, thereby inhibiting complement activity. Similarly, a decrease in the activity of a component of the complement system in the presence of an agent of interest (for example a sand fly salivary gland polypeptide, such as residues 21-139 of SEQ ID NO: 47, residues 18-446 of SEQ ID NO; 49, residues 23-115 of SEQ ID NO: 55, or residues 19-399 of SEQ ID NO: 63, or a variant thereof), indicates that the agent inhibits a component of the complement system, thereby inhibiting complement activity. In one specific embodiment, a decrease in hemolytic activity indicates that the agent inhibits a component of complement, such as (but not limited to) C2, C4, C4b, C2a, C3, C3b, or C5. In another specific embodiment, a decrease in hemolytic activity indicates that the agent affects C3b activity.

In one embodiment, the inhibition of the classical pathway by the sand fly anti-complement polypeptides disclosed herein can be measured by incubating human plasma and the sand fly anti-complement polypeptides, in the presence and absence of an agent, with an immune complex generated on a surface and then monitoring for the production of complement components. The plasma samples can be analyzed for the production and quantitation of complement components.

The sand fly anti-complement polypeptides described herein also can be evaluated in a variety of cell-based assays and animal models of complement- associated diseases or disorders. For example, efficacy in the prevention and/or treatment of arthritis can be evaluated in a collagen-induced arthritis model (Terato et al. Brit. J. Rheum. 35:828-838 (1966)). Potential arthritis prophylactics/therapeutics can also be screened in a model of antibody-mediated arthritis induced by the intravenous injection of a cocktail of four monoclonal antibodies, as described by Terato et al., J. Immunol. 148:2103-8 (1992); Terato et al., Autoimmunity 22:137-47 (1995). Candidates for the prevention and/or treatment of arthritis can also be studied in transgenic animal models, such as, for example, TNF-α transgenic mice (Taconic). These animals express human tumor necrosis factor (TNF-α), a cytokine which has been implicated in the pathogenesis of human rheumatoid arthritis. The expression of TNF- α. in these mice results in severe chronic arthritis of the forepaws and hind paws, and provides a simple mouse model of inflammatory arthritis.

EXAMPLES

Example 1

Discovery of anti-complement factors in the salivary glands of the sand fly

Lutzomyia longipalpis

This example is provided to show that a salivary gland polypeptide of Lutzomyia longipalpis (a New World sand fly and the main vector of visceral leishmaniasis) is a surprisingly potent anti-complement polypeptide. In order to identify the factor(s) involved in the blockade of complement cascade, several recombinant proteins from Lu. longipalpis salivary glands were generated. Recombinant Lu. longipalpis salivary proteins (LuIoSP) were expressed via a mammalian expression system and contained an His-tag extension in their C terminus. LJM19 (residues 23-115 of SEQ ID NO: 55), which has no homology reported in GenBank, was found to have potent anti-complement activity, acting on the classical pathway. This protein has no structure or motif that would otherwise have identified it as a having anti-complement activity.

The complement is a very important first line of defense against pathogens and is involved in many pathologies and syndromes. Lu. longipalpis salivary gland proteins identified herein as anti-complement factors can be envisioned to fight among others: complications during cardio-pulmonary surgeries, complications after hemodialysis, treatment of lupus erythematosus, treatment of juvenile arthritis or human rheumatoid arthritis, and other pathologies where alternative and/or classical pathways of complement are involved. Thus, LJM19 is a useful molecule to inhibit complement in vivo or can be used to design specific inhibitors of complement.

Sandflies and preparation of SGH - Lutzomyia longipalpis, Jacobina strain, were reared using as larval food a mixture of fermented rabbit feces and rabbit food. Adult sand flies were offered a cotton swab containing 20% sucrose and females were used for dissection of salivary glands at 4-7 days following emergence. Salivary glands were stored in groups of 10 pairs in 10 μl NaCl (150 mmol I^"1), Hepes buffer (10 mmol I^"1, pH 7.4) at -70⁰C until needed. Salivary glands were disrupted by ultrasonication within 1.5-mL conical tubes. Tubes were centrifuged at 10,000 g for 2 min and the resultant supernatant diluted in PBS and used for injections.

Cloning of Lu. longipalpis cDNA in His-tagged TOPO vector - VR2001-TOPO is a topoisomerase adaptation of VR1020 plasmid (Vical, Inc) described in a previous report (Oliveira et al, Vaccine, 24:374-90,2006). cDNA of each individual LuIoSP were amplified by PCR using a specific forward primer deduced from the amino- terminus region and a specific reverse primer containing an

ATGATGATGATGATGATG (SEQ ID NO: 71) motif between the stop codon and the carboxyterminus region. The expected amplified sequences were predicted to code for proteins starting after the natural cleavage site and containing a 6x His motif at the C-terminus region. PCR amplification conditions were: 1 hold of 94°C for 5 minutes, 2 cycles of 94°C for 30 seconds, 48°C for 1 minute, 72°C for 1 minute, 23 cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and 1 hold of 72°C for 7 minutes. Amplified products were extracted from a 1.0% agarose gel using Ultrafree-MC extraction kit (Millipore). 3 μL of each PCR product was immediately incubated with 0.5 μL, of VR2001-TOPO, 1 μL of salt solution (1.2 M NaCl, 0.06 M MgCl₂) and 1.5 μl H₂O for 5 minutes at room temperature. Transformation and selection of positive clones by sequencing were performed following standard procedures.

Expression and purification of Lu. longipalpis salivary proteins (LuIoSP) - For expression and purification of LuIoSP, an aliquot of a glycerol stock was added to 800 ml of LB/kanamycin (100 μg/ml) and incubated overnight on a shaker at 37°C. Plasmid purification was performed using the GenElute™ HP Endotoxin- Free Plasmid Megaprep Kit (Sigma- Aldrich, St. Louis, MO) following manufacturer's specifications. After elution, the plasmid was transferred to a Centricon plus-20 (Millipore, Bedford, MA) with a 10OkDa cutoff. The sample was washed three times with Ultrapure Water (KD Medical, Columbia, MD) and once with PBS Ix (9.0 g/1 NaCl, 232 mg/L KH₂PO₄, 703 mg/1 Na₂HPO₄, pH=7.2) (KD Medical, Columbia, MD) and purified through a 0.2 μM filter unit (Millipore, Bedford, MA). Recombinant proteins were produced by transfecting FreeStyle™ 293-F cells (Invitrogen, Carlsbad, CA) with 70μg of purified plasmid following the manufacturer's recommendations (Invitrogen, Carlsbad, CA). After 72 hours, transfected cell cultures were harvested and the supernatant filtered through a 0.45 μM filter unit and concentrated to 15 ml in an Amicon concentrator device (Millipore Corp., Bedford, MA, USA) in the presence of Buffer A (20 mM NaH₂PO₄, 20 mM Na₂HPO₄, pH 7.4 and 500 mM NaCl). A HiTrap™Chelating HP column (GE Healthcare) was charged with 5 ml Of Ni₂SO₄0.1M and washed with 10 ml of Milli-Q water and 30 ml of Buffer A using a vacuum manifold (Alltech associates Inc., Deerfield, IL).

The concentrated recombinant protein was then added to the HiTrap Chelating HP column using a vacuum manifold (Alltech associates, Inc, Deerfield, IL). The column was then connected to a Summit station HPLC system (Dionex, Sunnyvale, CA) consisting of a P680 HPLC pump and a PDA-100 photodiode array detector. The column was equilibrated for 30 minutes with Buffer A at 1 ml/min and following equilibration of baseline the following gradient was used to elute the protein: minute 0-10, 100% Buffer A; minute 10-20, 0% Buffer A, 100% Buffer B (20 mM NaH₂PO₄, 20 mM Na₂HPO₄. pH 7.4, 500 mM NaCl and 50 mM imidazole); minute 20-30 100% Buffer B; minute 30-60 a gradient of 100% Buffer B to 100% Buffer C (20 mM NaH₂PO₄, 20 mM Na₂HPO₄. pH 7.4, 500 mM NaCl and 500 mM imidazole); minute 60-70 100% Buffer C. Eluted proteins were detected at 280 nm and the eluted fractions were collected every minute on a 96 well microtiter plate using a Foxy 200 fraction collector.

An aliquot (5 μL) of all fractions were blotted on a nitrocellulose paper and the blot was blocked with 5% milk for 1 hour and incubated for 1 hour with polyclonal anti-saliva antibodies (either against total salivary protein and against the specific polypeptide) and 1 hour with anti-mouse Ap conjugated secondary antibody. Positive fractions were developed with WesternBlue® stabilized substrate for alkaline phosphatase (Promega, Madison, WI). An aliquot (5 μL) of positive fractions were run on SDS and silver stained using SilverQuest™ (Invitrogen). Imidazole was removed from the positive fractions by dialysis against PBS, pH 7.4.

Polyclonal antibodies against LuIoSP - VR2001- TOPO plasmids containing coding sequences of LuIoSP without His-tag extension were used to inject mice and generate polyclonal antibodies. Pre-immune samples were taken before the first injection and immune serum samples were taken after three injections given in two week intervals. Each mouse serum sample was pooled for experimentation.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blotting - The samples were treated with 4x NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA) and analyzed in NuPAGE 4-12% Bis Tris gels (Invitrogen) with NuPAGE MES SDS running buffer (Invitrogen, Carlsbad, CA). To estimate the molecular weight of the samples, SeeBlue® Plus2 marker from Invitrogen (myosin, phosphorylase, BSA, glutamic dehydrogenase, alcohol dehydrogenase, carbonic anhydrase, myoglobin red, lysozyme, aprotinin and insulin B chain) was used. The proteins in the gel were transferred to nitrocellulose membrane using iBlot™ device (Invitrogen, Carlsbad, CA). After blocking with 5 % milk in Tris buffered saline (TBS) with 0.1 % Tween-20 (TBS-T), pH 8.0, the membrane was incubated with sera of mice immunized with LuIoSP cDNA (1/100 in TBS-T 5 % milk). After two washes with TBS-T, the membrane was incubated with alkaline phosphotase-conjugated goat anti-mouse IgG (ZyMax™, Invitrogen) at 1/10000 in TBS-T 5 % milk for 40 minutes at room temperature. After three washes with TBS-T, the blots were developed by addition of Western Blue® stabilized substrate for alkaline phosphatase (Promega, Madison, WI).

Reagents and purified proteins for complement assays - Buffers used were: veronal buffered saline (VBS), 5 mM veronal, 145 mM NaCl, 0.02 % NaN₃, pH 7.3; GVB, VBS containing 0.1% gelatin; GVB++, GVB containing 0.15 mM CaCl₂ and 0.5 mM MgCl₂; GVBE, GVB containing 10 mM EDTA; and MgEGTA, 0.1 M MgCl₂, 0.1 M EGTA pH 7.3. C5-depleted serum was prepared by immunoadsorption of serum on anti-C5 sepharose (Morgan, Methods MoI Biol, 150:61-71, 2000). Complement proteins Factor H (Pangburn et al, J Exp Med, 146:257-70, 1977), C3 (Pangburn, J Immunol Methods, 102:7-14,1987; Hammer et al., J Biol Chem, 256:3995-4006,1981), Factor B (Gotze et al, J Exp Med, 134:90s-108s, 1971), and Factor D (Lesavre et al, J Immunol, 123:529-34, 1979) were all purified from normal human plasma as described in the references cited above. Cobra venom factor (CVF) was purified as previously described (Vogel et al, J Biol Chem, 257:8292-9, 1982). All proteins were stored at -75°C in VBS. Factor Band C3 (50- 100 μg) were radiolabeled with 500 μCi of ¹²⁵I for 30 minutes at 0⁰C in a glass tube coated with lodogen (Pierce Chemical Co, Rockford, IL). After incubation, the free ¹²⁵I was removed by centrifugal desalting through a G25 column pre-equilibrated with GVB (Christopherson, Methods Enzymol, 91:278-81, 1983). Specific activities for ¹²⁵I-labeled proteins ranged from 3 to 4 μ Ci / μg. Complement consumption assays - This test is based on the complement consuming effect of cobra venom factor (CVF), a protein known to consume complement activity. If a CVF containing sample is incubated with human serum, the complement proteins are consumed depending on the CVF activity. The remaining complement activity of the serum can be detected subsequently using sensitized sheep erythrocytes. Thus, various LuIoSP (LJM04, LJM17, LJM19, LJMIl and LJM26), at 0.3 - 0.4 μM final concentration, were each incubated with NHS (normal human serum; 40% final) for up to 60 minutes at 37°C. CVF was used as a positive control. Remaining complement activity was assayed by adding 10 μl of the mix to 40 μl of rabbit erythrocytes (E_R; 3 x 10⁶ cells) in GVB with MgEGTA (5 mM final), for 20 min at 37°C. To determine the extent of hemolysis, 230 μl cold GVBE was added, the samples were centrifuged, and the optical density of supernatant was determined at 414 nm. The percent lysis was determined by subtracting the A₄₁₄ in the absence of serum, and dividing by the maximum possible A₄₁₄ determined by water lysis of the erythrocytes.

The percent hemolysis of samples incubated for up to 60 minutes with LJM04, LJM17, LJM19, LJMI l or LJM26 was similar to that of the GVB negative control sample (Figure IA). In contrast, the sample incubated with cobra venom factor (CVF) protein, which is known to consume complement activity, exhibited a complete depletion of complement by 30 minutes, as demonstrated by a total abrogation of hemolysis. Thus, none of the LuIoSP consumed complement. This confirmed that these proteins were not activators of the complement cascade and validated the use of the LuIoSP in further in vitro inhibition assays of complement cascade.

Hemolytic assays - Inhibition of alternative pathway-mediated lysis of E_R by LuIoSP was measured by mixing, on ice, GVB, NHS (15% final), and 0.1 M MgEGTA (5 mM final concentration) in the presence of 0.6-0.8 μM LuIoSP or 10 mM EDTA. E_R (1 X 10⁶ cells) were added and the mix (20 μl total) was transferred to a 37°C water bath and incubated for 20 minutes. To determine the extent of hemolysis, 100 μl cold GVBE was added, the samples were centrifuged and the optical density of supernatant was determined at 414 nm. The percent lysis was determined by subtracting the A₄₁₄ in the absence of serum, and dividing by the maximum possible A_4I4 determined by water lysis of the erythrocytes. Samples containing LJM04, LJMIl and LJM26 only exhibited 10%, 1%, and 1% lysis of rabbit erythroctyes, respectively, compared to 100% lysis by samples containing LJM19, LJM17, and buffer alone. Thus, LJM04, LJMIl, and LJM26 demonstrated 90%, 99% and 99% inhibition (at 0.3-0.4 μM), respectively, of the alternative pathway of complement (Figure IB).

Inhibition of classical pathway-mediated lysis of antibody-coated sheep erythrocytes (EA) by LuIoSP was measured by mixing, on ice GVB++, NHS (1 % final), in the presence of 0.3-0.4 μM LuIoSP or 10 mM EDTA. EA (1 x 10⁷ cells) were added and the mix was transferred to a 37°C water bath and incubated for 30 minutes. The percent lysis was determined as described above. Inhibition of classical pathway (CP)-mediated lysis of antibody-coated sheep erythrocytes (EA) was tested for each individual LuIoSP.

In one experiment, alternative pathway inhibitors LJM04, LJMI l and LJM26 also inhibited the classical pathway of complement, demonstrating 90%, 99%, and 95% inhibition (at 0.3-0.4 μM), respectively, compared to buffer alone (Figure 1C). In addition, LJM19, despite no activity in inhibiting the alternative pathway, inhibited the classical pathway of complement (0% cell lysis, thus 100% of inhibition at 0.3-0.4 μM) (Figure 1C). LJM17 showed no inhibitory activity of complement cascade (Figure 1C). However, in subsequent assays measuring inhibition of classical pathway (CP)-mediated lysis, only LJM19 consistently demonstrated almost complete (98%) inhibition of the classical complement pathway (Figure 2). Thus, LJM19 is specific to the classical pathway of complement and demonstrates a surprisingly potent anti-complement activity, compared to other Lu. longipalpis salivary gland polypeptides.

Measurement-of Inhibition of C3b deposition - To further identify the mechanism of inhibition of LuIoSP targeting both pathways (LJM04, LJMIl and LJM26), deposition of C3b on zymosan (Sigma- Aldrich, St. Louis, MO) was tested using purified C3, factor B and factor D as previously described (Rawal et ah, J Bioi Chem, 273:16828-16835, 1998) with the substitution of nickel to stabilize the C3 convertases on the surface of the cells (Fishelson et ah, J Immunol, 129:2603-2607, 1982). The number of bound C3b molecules was determined to be ~115,000 per zymosan particle by radiolabeled factor Bb binding (Rawal et al, supra). Deposition of C3b on E_R was measured by mixing, on ice, GVB, C5-depleted serum (10% final), ¹²⁵I-C3, 2.5 mM MgEGTA, and the LuIoSP being tested. The mixture was transferred to a 37°C water bath for 15 minutes. The cells were sedimented rapidly (2 minutes, 10,000 x g) through 250 μl of 20% sucrose in GVBE in a microfuge tube to separate bound from free radiolabel. The bottoms of the tubes were cut off and the radioactivity in the cell pellet and the supernatant were measured to determine the percent ¹²⁵I-C3b bound. LJM04, UMl 1 and LJM26 specifically inhibit C3b deposition demonstrating 75%, 99% and 99% of inhibition (at 0.3-0.4 μM), respectively, compared to buffer alone (Figure ID).

C3/C5 Convertase Decay Acceleration Assays - Decay accelerating activity expressed by LuIoSP or factor H was assessed by determining their ability to accelerate the natural release of ¹²⁵I- labeled Bb from zymosan-bound C3b,Bb. The C3b,Bb complexes were formed by incubating 2 x 10⁷ ZymC3b with 0.3 μg (-0.8 μCi) ¹²⁵I-factor B and 0.4 μg factor D in 40 μl GVB containing 1.4 mM NiCl₂ at 22°C for 3 minutes. Formation of the C3 convertase was stopped by the addition of 160 μl of GVBE. The ZymC3b, ¹²⁵I-Bb particles (10 μl) were added immediately to reaction mixtures containing 0.3-2.7 μM factor H or 0.3-0.4 μM LuIoSP, in 10 μl GVBE. After 15 minutes at 22°C the cells were sedimented rapidly (2 minutes, 10,000 x g) through 250 μl of 20% sucrose in GVBE in a microfuge tube. The bottoms of the tubes were cut off and the radioactivity in the cell pellet and the supernatant were measured to determine the percent Bb remaining bound. As shown in Figure IE, none of the LuIoSP proteins accelerate the decay of C3b,Bb convertase, despite the fact it is the usual mechanism of alternative pathway inhibition. BIAcore binding assays have been performed (data not shown). Because LJM04, LJMIl and LJM26 could inhibit the pre-assembled AP C3/C5 convertase, the binding of the proteins to C3b attached (by standard amine coupling) to a BIAcore chip was examined. LJM04, LJMIl and LJM26 showed evidence of direct binding to C3b (data not shown). Kd values were estimated in the range of 50 nM, which is a high affinity. Thus, LJM04, LJMIl and LJM26 can be envisioned as new tools for understanding the physiological and pathophysiological mechanisms of C3b molecule.

Example 2

Method of treating a subject with sand fly salivary gland anti-complement polypeptides

This example describes a protocol to prevent or treat a disease or pathology associated with complement activation in a subject using the sand fly salivary gland anti-complement polypeptides described herein, or a polynucleotide encoding the polypeptide, for example, a Lu. longipalpis salivary gland polypeptide, such as SEQ ID NO: 47, residues 21-139 of SEQ ID NO: 47, SEQ ID NO: 49, residues 18-446 of SEQ ID NO: 49, SEQ ID NO: 55, residues 23-115 of SEQ ID NO: 55, SEQ ID NO: 63, or residues 19-399 of SEQ ID NO: 63, or variants or fragments thereof, or a polynucleotide, such as SEQ ID NO: 48, residues 40-456 of SEQ ID NO: 48, residues 100-456 of SEQ ID NO: 48, SEQ ID NO: 50, residues 96-1616 of SEQ ID NO: 50, residues 147-1616 of SEQ ID NO: 50, SEQ ID NO: 56, residues 16-360 of SEQ ID NO: 56, residues 82-360 of SEQ ID NO: 56, SEQ ID NO: 64, residues 20- 1216 of SEQ ID NO: 64, or residues 74-1216 of SEQ ID NO: 64, or a degenerate variant thereof. Such diseases or pathologies include (but are not limited to) septic shock, complement activation during cardiopulmonary bypass surgery (due, for example, to interaction of blood with the extracorporeal circuit of the heart- lung machine used during such surgeries), systemic lupus erythematosus (SLE) (lupus nephritis and resultant glomerulonephritis and vasculitis), rheumatoid arthritis (RA), juvenile chronic arthritis, adult respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, pemphigus, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulemia, antiphospholipid syndrome, age- related macular degeneration, uveitis, diabetic retinopathy, allotransplantation, hemodialysis, chronic occlusive pulmonary disetress syndrome (COPD), asthma, and aspiration pneumonia, inflammatory bowel disease (IBD), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), transplantation associated diseases including graft rejection and graft- versus host disease as well as other pathologies and diseases, are believed to have a complement-mediated component. This protocol is intended to serve as an example of such a treatment method, and is not meant to be limiting. Those of skill in the art will be able to modify the protocol to suit the needs of the subject, and to optimize for the particular compounds used. Subjects can, but need not, have received previous therapeutic treatments.

A sand fly salivary gland anti-complement polypeptide or poly nucleotide is administered orally or parenterally in dosage unit formulations containing standard, well known non-toxic physiologically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intra- arterial injection, or infusion techniques. The sand fly salivary gland anti-complement polypeptides can be administered in dosages, as described above, depending on the polypeptide or polynucleotide used. Therapeutically effective doses of sand fly salivary gland anti-complement polypeptides or polynucleotides can be delivered to the subject before, after or concurrently with the other anti-complement agents.

A typical treatment course can comprise about six doses delivered over a 7 to 21 day period. Upon election by the clinician, the regimen can be continued six doses every three weeks or on a more frequent (daily, twice daily, four times a day, etc.) or less frequent (monthly, bimonthly, quarterly, etc.) basis. Of course, these are only exemplary times for treatment, and the skilled practitioner will readily recognize that many other time-courses are possible. The sand fly salivary gland anti-complement polypeptides can be combined with any of a number of conventional treatment regimens. Regional delivery of sand fly salivary gland anti- complement polypeptides is an efficient method for delivering a therapeutically effective dose to counteract the clinical disease. Therapeutically effective doses of sand fly salivary gland anti-complement polypeptides or polynucleotides can be delivered to the subject before, after or concurrently with the other anti-complement agents.

Clinical responses can be defined by an acceptable measure. For example, a complete response can be defined by the disappearance of all measurable disease (as measured, for example, by decreased cell lysis, decreased complement activity, decreased symptoms of disease) for at least a month. A partial response can be defined by a 50% or greater reduction of all measurable disease.

Of course, the above-described treatment regimes can be altered by those of skill in the art, who will be able to take the information disclosed in this specification and optimize treatment regimes.

Example 3

Inhibition of Leishmania chagasi parasite load with sand fly anti-complement polypeptide anti-sera

Balb/c mouse blood is collected by cardiac draw using a ImI syringe and 25Ga. 5/8" needle coated in heparin. The blood is centrifuged at 2,000 RPM for 10 minutes at room temperature and the serum removed for heat inactivation by incubation at 56°C for 1 hour. An equivalent amount of the removed serum is pooled from three mice immunized four times with a sand fly salivary gland anti- complement polypeptide and heat inactivated at 56°C for 1 hour. The blood cells were washed twice by centrifugation with phosphate buffered saline (PBS), pH 7.2, to removed residual complement and stored at 4°C until used.

Leishmania chagasi are grown at 26°C in Schneider's Drosophila medium (Invitrogen) with 20% heat-inactivated fetal bovine serum and penicillin- streptomycin-glutamine (100U-100μg-292ng). Culture media is added to log-phase growth cultured parasites 1 day before collection to enrich for procyclic promastigotes. The culture is then centrifuged at 19 x g for 3 minutes to remove large clumps of parasites and debris. The supernatant is then centrifuged at 1,500 RPM for 10 minutes at 22°C, washing the parasites once with PBS, prior to counting parasite concentration using a C-Chip hemocytometer. The heat inactivated serum (from immunized and control mice) and washed blood cells are mixed with 2xlO⁶/ml L. chagasi and placed in the glass feeding chamber covered with the chick skin membrane. The feeding chamber is heated by a circulating water bath set to 38⁰C and the sand flies are allowed to feed in the dark for 3 hours. Fully engorged sand flies are separated six hours after feeding. At 6 hours and 1, 2, 3, 4 and 6 days after blood feeding the midgut of sand flies are dissected in PBS and collected in pools of 10 before homogenization in lOOμl of PBS using a pestle and small tissue grinder. The number of leishmania parasites in lOμl of midgut homogenate diluted 1:2, 1:5 or 1:10 in PBS 0.2% formalin is assessed by microscopy using a C-Chip hemocytometer. On day 12 after blood feeding individual midgut are dissected and homogenized to quantify the parasite load. Parasite load in sand flies which feed on a serum samples from immunized mice is reduced, compared to the parasite load in sand flies which feed on a serum samples from control mice.

Example 4 Production of a Transmission Blocking Vaccine in Dogs

Dogs infected with leishmania (which are either asymptomatic or symptomatic) are immunized with 100 μg of recombinant sand fly salivary gland anti-complement polypeptide (for example, SEQ ID NO: 47, residues 21-139 of SEQ ID NO: 47, SEQ ID NO: 49, residues 18-446 of SEQ ID NO: 49, SEQ ID NO: 55, residues 23-115 of SEQ ID NO: 55, SEQ ID NO: 63, or residues 19-399 of SEQ ID NO: 63) three times at 21 day intervals. The recombinant polypeptide is injected in combination with an adjuvant, such as alum or titermax. Control dogs (infected with leishmania and which are either asymptomatic or symptomatic) are immunized with adjuvant in the absence of the sand fly salivary polypeptide. In some experiments, blood is withdrawn from the control and experimental dogs following one or more immunizations and antibody titer against the sand fly salivary gland polypeptide is measured. Dogs with measurable antibody titers and the control dogs are exposed to sand flies. Fully engorged sand flies from the experimental and control groups are separated after feeding. A portion of the experimental and control group sand flies are dissected to analyze parasite load, whereas the remaining are saved for future exposure to uninfected animals.

The midgut of sand flies are dissected in PBS and collected before homogenization in PBS using a pestle and small tissue grinder. The number of leishmania parasites in lOμl of midgut homogenate diluted 1:2, 1:5 or 1:10 in PBS 0.2% formalin is assessed by microscopy using a C-Chip hemocytometer. On day 12 after blood feeding individual midgut are dissected and homogenized to quantify the parasite load. Parasite load in sand flies which feed on dogs with antibody titers against the sand fly salivary anti-complement polypeptide (experimental group) is reduced, compared to the parasite load in sand flies which feed on control dogs.

An animal (for example, dogs, mice, hamsters, chimpanzees) uninfected with leishmania are exposed to the remaining sand flies from the control and experimental groups. After sufficient time for sand fly feeding, the dogs are tested for parasite load. Animals exposed to sand flies from the experimental group show no, or minimal, infection from leishmania, compared to animals exposed to sand flies from the control group.

In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of inhibiting a component of the classical complement pathway in a sample in vitro, comprising contacting the sample with an effective amount of a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55, thereby inhibiting the component of the classical complement pathway.

2. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55.

3. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55.

4. The method of claim 3, wherein the polypeptide comprises an amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55.

5. The method of claim 4, wherein the polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 55.

6. The method of claim 1, further comprising contacting the sample with a cell sample and measuring lysis of the cell sample.

7. The method of claim 1, wherein inhibiting a component of the classical complement pathway comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% inhibition, compared to a sample that has not been contacted with the polypeptide.

8. A method of preventing or treating a disorder associated with increased complement activation in a subject, comprising: selecting a subject with a disorder associated with increased complement activation administering to the subject a therapeutically effective amount of a polypeptide having at least 90% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55, thereby preventing or treating the disorder associated with increased complement activation in the subject.

9. The method of claim 8, wherein the polypeptide inhibits an activity of a classical pathway component of the complement system.

10. The method of claim 9, wherein the activity is cell lysis.

11. The method of claim 8, wherein treating the subject comprises inhibiting lysis of a cell sample obtained from the subject by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, compared to a subject who has not been administered a therapeutically effective amount of the polypeptide.

12. The method of claim 8, wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55.

13. The method of claim 12, wherein the polypeptide comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55.

14. The method of claim 13, wherein the polypeptide comprises an amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55.

15. The method of claim 14, wherein the polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 55.

16. The method of claim 8, wherein selecting the subject comprises selecting a subject having septic shock, complement activation during cardiopulmonary bypass surgery, lupus nephritis, glomerulonephritis, rheumatoid arthritis, juvenile chronic arthritis, adult respiratory distress syndrome, remote tissue injury after ischemia and reperfusion, pemphigus, cardioplegia- induced coronary endothelial dysfunction, type I or II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulemia, antiphospholipid syndrome, age-related macular degeneration, uveitis, diabetic retinopathy, allotransplantation, hemodialysis, chronic occlusive pulmonary distress syndrome, asthma, and aspiration pneumonia, inflammatory bowel disease, idiopathic inflammatory myopathies, graft rejection, or graft- versus host disease

17. The method of claim 8, wherein administering to the subject a therapeutically effective amount of a polypeptide comprises administering a nucleic acid sequence that encodes a polypeptide having at least 90% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55.

18. The method of claim 17, wherein the nucleic acid sequence comprises the sequence set forth as residues 82-360 of SEQ ID NO: 56, or a degenerate variant thereof.

19. The method of claim 18, wherein the nucleic acid comprises the sequence set forth as residues 16-360 of SEQ ID NO: 56, or a degenerate variant thereof.

20. The method of claim 19, wherein the nucleic acid is operably linked to an expression control sequence.

21. The method of claim 20, wherein the expression control sequence is a promoter.

22. The method of claim 21, wherein the promoter is an inducible or constitutive promoter.

23. A method for preventing the development of a Leishmania parasite in a sand fly, comprising: administering to a subject an immunologically effective amount of a polypeptide having at least 90% sequence identity to the amino acid sequence set forth as residues 23-115 of SEQ ID NO: 55, wherein antibodies produced in the subject specifically bind the polypeptide set forth as residues 23-115 of SEQ ID NO: 55, and wherein the antibodies, when ingested by the sand fly, prevent Leishmania development in the sand fly, thereby preventing the development of the Leishmania parasite in the sand fly.

24. The method of claim 23, wherein the subject is a dog or a human.