Peptide sequences and compositions

Abstract

Provided is a polypeptide composition comprising one or more polypeptides, which polypeptides are immunogenic in a vertebrate such that they cause the vertebrate to produce immune system cells capable of recognizing at least one epitope from an arthropod saliva protein fraction, wherein the arthropod saliva protein fraction has a mass of 40 kDA or less, and wherein the polypeptides are selected independently from: the polypeptide sequences of SEQ ID 1-44 or sub-sequences from these sequences, the sub-sequences having 7 amino acids or more; or from polypeptide sequences having 85% homology or more with one or more of the above sequences and contained in one or more of the following databases: GenBank, Protein Data Bank (PDB), SwissProt, Protein Information Resource (PIR), Protein Research Foundation (PRF), or CDS translations of these.

Claims

1. A polypeptide composition comprising .[.an.]. .Iadd.one or more .Iaddend.isolated .[.polypeptide.]. .Iadd.polypeptides and a carrier.Iaddend., the .Iadd.one or more .Iaddend.isolated .[.polypeptide being.]. .Iadd.polypeptides including a polypeptide of .Iaddend.SEQ ID NO: 35 or a .Iadd.polypeptide with a .Iaddend.sequence having 85% homology or more to SEQ ID NO.Iadd.: .Iaddend.35, wherein the .[.isolated polypeptide elicits an immune response in vertebrates against at least one epitope of a protein from an arthropod saliva fraction, wherein the arthropod saliva protein fraction has a mass of 40 kDa or less.]. .Iadd.polypeptide composition elicits an immune response in vertebrates against a protein comprising SEQ ID NO: 35.Iaddend..

2. The polypeptide composition according to claim 1, wherein the .[.composition further comprises one or more further isolated polypeptides, the one or more further isolated polypeptides being.]. .Iadd.one or more isolated polypeptides further comprise a polypeptide of SEQ ID NO: 6, .Iaddend.SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a polypeptide .Iadd.with a sequence .Iaddend.having 85% homology or more to .Iadd.SEQ ID NO:6, .Iaddend.SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

.[.3. The polypeptide composition according to claim 1, which composition comprises 2 or more isolated polypeptides..].

.[.4. The polypeptide composition according to claim 1, wherein the protein from the arthropod saliva fraction has a mass from 20 kDa to 40 kDa, or has a mass of 20 kDa or less..].

.[.5. The polypeptide composition according to claim 1, wherein the isolated polypeptide includes a cytotoxic T lymphocyte (CTL) epitope, and/or the isolated polypeptide includes a T helper lymphocyte (Th) epitope, and/or the isolated polypeptide includes a B lymphocyte epitope..].

.[.6. The polypeptide composition according to claim 1, comprising 2, 3, 4, 5, 6 or more epitopes..].

.[.7. The polypeptide composition according to claim 1, which is immunogenic against a plurality of arthropod salivary proteins..].

.[.8. The polypeptide composition according to claim 1, wherein the arthropod saliva protein fraction is a mosquito saliva protein fraction from any mosquito species..].

.[.9. The polypeptide composition according to claim 1, further comprising one or more further sequences from an arthropod salivary protein..].

10. The polypeptide composition according to claim 2, .[.in which.]. .Iadd.wherein .Iaddend.the one or more further isolated polypeptides .[.present are.]. .Iadd.further comprise: a) .Iaddend.SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.Iadd.; b) SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32; c) SEQ ID NO: 91, SEQ ID NO: 115 or SEQ ID NO: 116; d) SEQ ID NO: 91, SEQ ID NO: 115 and SEQ ID NO: 116; e) SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117; f) SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117; g) SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 115, or SEQ ID NO: 116; or h) SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 115, and SEQ ID NO: 116.Iaddend..

.[.11. The polypeptide composition of claim 1, further comprising a pharmaceutically acceptable carrier..].

.[.12. A method of producing a polypeptide composition of claim 1, which method comprises mixing or combining one or more of the polypeptides with one or more further polypeptides, and/or with one or more further components, such as a carrier, an excipient, an adjuvant, a buffer, or a stabiliser..].

13. A polypeptide construct comprising an isolated polypeptide .Iadd.according to claim 1 .Iaddend.linked to a carrier.[., the isolated polypeptide being SEQ ID NO: 35 or a sequence having 85% homology or more to SEQ ID NO 35, wherein the isolated polypeptide elicits an immune response in vertebrates against at least one epitope of a protein from an arthropod saliva fraction, wherein the arthropod saliva protein fraction has a mass of 40 kDa or less.]..

.[.14. A method of producing a polypeptide construct as defined in claim 13, which method comprises attaching, combining or mixing a polypeptide composition with the carrier..].

15. The polypeptide construct according to claim 13, wherein the carrier comprises an adjuvant and/or an excipient.

16. The polypeptide composition according to claim 2, wherein the one or more .[.further.]. isolated polypeptides include .Iadd.a polypeptide having 85% homology or more to SEQ ID NO:6, .Iaddend.a polypeptide having 85% homology or more to SEQ ID NO: 20, a polypeptide having 85% homology or more to SEQ ID NO: 28, a polypeptide having 85% homology or more to SEQ ID NO: 30, a polypeptide having 85% homology or more to SEQ ID NO: 31, .[.or.]. a polypeptide having 85% homology or more to SEQ ID NO: 32.Iadd., SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116 and/or SEQ ID NO: 117.Iaddend..

.[.17. The polypeptide composition according to claim 3, which composition comprises from 2 to 12 isolated polypeptides..].

18. The polypeptide composition according to claim .[.3.]. .Iadd.1.Iaddend., .[.which composition comprises from 2-6 isolated polypeptides.]. .Iadd.wherein the one or more isolated polypeptides includes a polypeptide having 85% homology or more to SEQ ID NO: 35 or SEQ ID NO: 120.Iaddend..

19. The polypeptide composition according to claim .[.8.]. .Iadd.2.Iaddend., wherein the .[.mosquito species is Anopheles gambiae.]. .Iadd.one or more isolated polypeptides further comprise: a) SEQ ID NO: 91, SEQ ID NO: 115, SEQ ID NO: 116 or SEQ ID NO: 120; b) SEQ ID NO: 91, SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 120; c) SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 120; d) SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 120; e) SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 115, SEQ ID NO: 116 or SEQ ID NO: 120; or f) SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 120.Iaddend..

.Iadd.20. The polypeptide construct according to claim 1, wherein the carrier comprises an adjuvant and/or an excipient..Iaddend.

.Iadd.21. A polypeptide composition comprising one or more isolated polypeptides and a carrier, the one or more isolated polypeptides including a polypeptide of SEQ ID NO: 91 or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 91, wherein the polypeptide composition elicits an immune response in vertebrates against a protein comprising SEQ ID NO: 91..Iaddend.

.Iadd.22. The polypeptide composition according to claim 21, wherein the sequence having 85% homology or more to SEQ ID NO: 91 is SEQ ID NO: 6..Iaddend.

.Iadd.23. The polypeptide composition according to claim 21, wherein the one or more isolated polypeptides further comprise a polypeptide of SEQ ID NO: 115, SEQ ID NO: 116 or SEQ ID NO: 120, or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 115, SEQ ID NO: 116 or SEQ ID NO: 120..Iaddend.

.Iadd.24. The polypeptide composition according to claim 21, wherein the carrier comprises an adjuvant and/or an excipient..Iaddend.

.Iadd.25. The polypeptide composition according to claim 23, comprising SEQ ID NO: 91, SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 120..Iaddend.

.Iadd.26. The polypeptide composition according to claim 23, comprising a polypeptide having 85% homology or more to SEQ ID NO: 91, a polypeptide having 85% homology or more to SEQ ID NO: 115, a polypeptide having 85% homology or more to SEQ ID NO: 116 and a polypeptide having 85% homology or more to SEQ ID NO: 120..Iaddend.

.Iadd.27. The polypeptide construct comprising an isolated polypeptide according to claim 21 linked to a carrier..Iaddend.

.Iadd.28. A polypeptide composition comprising one or more isolated polypeptides and a carrier, the one or more isolated polypeptides including SEQ ID NO: 115 or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 115, wherein the polypeptide composition elicits an immune response in vertebrates against a protein comprising SEQ ID NO: 115..Iaddend.

.Iadd.29. The polypeptide composition according to claim 28, wherein the sequence having 85% homology or more to SEQ ID NO: 115 is SEQ ID NO: 30..Iaddend.

.Iadd.30. The polypeptide composition according to claim 28, wherein the one or more isolated polypeptides further comprise a polypeptide of SEQ ID NO: 91, SEQ ID NO: 116 or SEQ ID NO: 120, or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 91, SEQ ID NO: 116 or SEQ ID NO: 120..Iaddend.

.Iadd.31. The polypeptide construct according to claim 28, wherein the carrier comprises an adjuvant and/or an excipient..Iaddend.

.Iadd.32. The polypeptide composition according to claim 30, wherein the one or more isolated polypeptides present are SEQ ID NO: 91, SEQ ID NO: 116 and SEQ ID NO: 120..Iaddend.

.Iadd.33. The polypeptide composition according to claim 30, wherein the one or more isolated polypeptides include a polypeptide having 85% homology or more to SEQ ID NO: 91, a polypeptide having 85% homology or more to SEQ ID NO: 116 and a polypeptide having 85% homology or more to SEQ ID NO: 120..Iaddend.

.Iadd.34. The polypeptide composition comprising an isolated polypeptide according to claim 28 linked to a carrier..Iaddend.

.Iadd.35. A polypeptide composition comprising one or more isolated polypeptides and a carrier, the one or more isolated polypeptides including a polypeptide of SEQ ID NO: 116 or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 116, wherein the polypeptide composition elicits an immune response in vertebrates against a protein comprising SEQ ID NO: 116..Iaddend.

.Iadd.36. The polypeptide composition according to claim 35, wherein the sequence having 85% homology or more to SEQ ID NO: 116 is SEQ ID NO: 31..Iaddend.

.Iadd.37. The polypeptide composition according to claim 35, wherein the one or more isolated polypeptides further comprise a polypeptide of SEQ ID NO: 91, SEQ ID NO: 115 or SEQ ID NO: 120, or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 91, SEQ ID NO: 115 or SEQ ID NO: 120..Iaddend.

.Iadd.38. The polypeptide construct according to claim 35, wherein the carrier comprises an adjuvant and/or an excipient..Iaddend.

.Iadd.39. The polypeptide composition according to claim 37, in which the one or more isolated polypeptides present are SEQ ID NO: 91, SEQ ID NO: 115 and SEQ ID NO: 120..Iaddend.

.Iadd.40. The polypeptide composition according to claim 37, wherein the one or more isolated polypeptides include a polypeptide having 85% homology or more to SEQ ID NO: 91, a polypeptide having 85% homology or more to SEQ ID NO: 115 and a polypeptide having 85% homology or more to SEQ ID NO: 120..Iaddend.

.Iadd.41. The polypeptide construct comprising an isolated polypeptide according to claim 35 linked to a carrier..Iaddend.

.Iadd.42. A polypeptide composition comprising one or more isolated polypeptides and a carrier, the one or more isolated polypeptides including a polypeptide of SEQ ID NO: 120 or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 120, wherein the polypeptide composition elicits an immune response in vertebrates against a protein comprising SEQ ID NO: 120..Iaddend.

.Iadd.43. The polypeptide composition according to claim 42, wherein the sequence having 85% homology or more to SEQ ID NO: 120 is SEQ ID NO: 35..Iaddend.

.Iadd.44. The polypeptide composition according to claim 42, wherein the one or more isolated polypeptides further comprise a polypeptide of SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116 or SEQ ID NO: 117, or a polypeptide with a sequence having 85% homology or more to SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116 or SEQ ID NO: 117..Iaddend.

.Iadd.45. The polypeptide construct according to claim 42, wherein the carrier comprises an adjuvant and/or an excipient..Iaddend.

.Iadd.46. The polypeptide composition according to claim 43, in which the one or more isolated polypeptides present are SEQ ID NO: 91, SEQ ID NO: 115 and SEQ ID NO: 116..Iaddend.

.Iadd.47. The polypeptide composition according to claim 43, in which the one or more isolated polypeptides present are SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117..Iaddend.

.Iadd.48. The polypeptide composition according to claim 43, wherein the one or more isolated polypeptides include a polypeptide having 85% homology or more to SEQ ID NO: 91, a polypeptide having 85% homology or more to SEQ ID NO: 115 and a polypeptide having 85% homology or more to SEQ ID NO: 116..Iaddend.

.Iadd.49. The polypeptide composition according to claim 43, wherein the one or more isolated polypeptides include a polypeptide having 85% homology or more to SEQ ID NO: 105, a polypeptide having 85% homology or more to SEQ ID NO: 113, a polypeptide having 85% homology or more to SEQ ID NO: 115, a polypeptide having 85% homology or more to SEQ ID NO: 116 and a polypeptide having 85% homology or more to SEQ ID NO: 117..Iaddend.

.Iadd.50. The polypeptide construct comprising an isolated polypeptide according to claim 42 linked to a carrier..Iaddend.

Description

(1) The present invention will be described in more detail by way of example only with reference to the following Figures, in which:

(2) FIGS. 1 and 9 show IEF gels of anopheles gambiae salivary glands (Comassie Blue stain), as produced in accordance with the protocols set out in the Examples;

(3) FIG. 2 shows an SDS-PAGE of the IEF gel of FIG. 1 (silver stain)the red rectangle indicates the location of one of the targeted proteins (<30 kDa)

(4) FIGS. 3A-3I show data on the effect of the vaccine on the fecundity of mosquitoes, as follows:

(5) TABLE-US-00008 3A: Percent that fed 3B: Mean no eggs produced 3C: Mean no eggs laid 3D: Percent hatch rate 3E: Mean no larvae 3F: Mean no pupae 3G: Percent pupation 3H: Percent emergence 3I: Mean no adults

(6) FIG. 4 shows the data on the percent survival of mosquitoes after feeding on immunized mice.

(7) FIG. 5 shows IFN-gamma production following 96 hour stimulation in vitro with the antigens in Experiment 3.

(8) FIG. 6 shows the total Ig response in sera to the antigens according to Experiment 3.

(9) FIG. 7 shows total Ig response in sera to the AGS-mix at day 21 in accordance with Experiment 4.

(10) FIG. 8 shows the AGS-mix immunised group of Experiment 4 displaying an increased survival rate as compared with those in the control NRP-mix immunised group.

(11) The polypeptide described above typically comprises one or more (preferably two or more) epitopes. These epitopes are preferably T cell epitopes, such as cytotoxic T lymphocyte (CTL) epitopes, but may also contain B cell epitopes. Generally the polypeptide is immunogenic to an arthropod saliva protein, and preferably to a plurality of such proteins. In the present context, a polypeptide immunogenic to an arthropod saliva protein is understood to mean a polypeptide that is part of an arthropod saliva protein and that elicits an immune system response. One method for determining whether a polypeptide possesses such immunogenicity is set out in Experiment 2 below. However, the present invention is not limited to such methods, and the skilled person may select any known method for determining immunogenicity, as desired.

(12) In the present invention, the polypeptide composition comprises one or more sequences as described above. Typically, two, three, four, five or more such sequences may be present in the polypeptide, if desired. The more such epitopes are present, the greater the breadth of protection afforded within a population of humans and/or animals individuals with differing HLAs or MHCs. This is particularly so if the epitopes included are from the saliva of a plurality of differing arthropods or shared by salivary proteins of different arthropod species, and can thus offer protection against diseases carried by a plurality of different arthropods. Typically the polypeptide composition comprises 10 polypeptides or fewer, preferably 6 polypeptides or fewer, and typically from 2-10 polypeptides, and more preferably from 2-6 polypeptides.

(13) The polypeptide composition according to the present invention may also comprise one or more further sequences that are not epitopes, if desired. Typically the further sequences are from one or more arthropod saliva proteins, preferably selected from the sequences of SEQ ID 45-85 or sub-sequences of these. These sequences may be situated between two or more of the sequences (the epitopes) described above, or may be situated at one or both ends of the polypeptide. The presence of such further sequences should not affect the function of the polypeptide, provided that the polypeptide as a whole does not become too large, interfering with the presentation of the epitopes in the vertebrate's immune system.

(14) In the most preferred embodiments, the further sequences from the above-mentioned proteins are ones that are (or are within) the following sequences:

(15) TABLE-US-00009 SEQID45->gi|18389913|gb|AAL68793.1|AF457563.sub.-1hypotheticalprotein16[Anopheles gambiae] MHLTLFTVAVLLLAAAALLLLLPPAYSTTLTPPAPPRLSHLGITIGRI SEQID46->gi|18389909|gb|AAL68791.1|AF457561.sub.-1hypotheticalprotein14[Anopheles gambiae] MPLSYCHLFLTHTLARALSFSRSDCLKFSEKRLLFSGSKTFPTTLL SEQID47->gi|18389907|gb|AAL68790.1|AF457560.sub.-1hypotheticalprotein13[Anopheles gambiae] MKNVFFALLLVVLVCCLVSVQGNEIIQNVVKRSIPLRQLILQHNALDDSNSDSGSQ SEQID48->gi|18389903|gb|AAL68788.1|AF457558.sub.-1hypotheticalprotein11[Anopheles gambiae] MCIFFQAGIKLLVLLICLFFYHTHCTTAYLWLAMGVEAKSIKARGTAHSKSRTSTN SEQID49->gi|62546227|gb|AAX86005.1|hyp3.5precursor[Anophelesgambiae] MFLKGSFPRFQMCVMLIGFFSSAKCLMCFADWEGMLLMTMEVFDFQLIVFTPVLKRS SEQID50->gi|18389899|gb|AAL68786.1|AF457556.sub.-1salivarygland7-likeprotein [Anophelesgambiae] MAGESQKNARSKQNDYQALLGLCCPWIDLAAADLPMRRHAKAREAINFLLQAHEA GPNEEPSLPA SEQID51->gi|18389911|gb|AAL68792.1|AF457562.sub.-1hypotheticalprotein15[Anopheles gambiae] MKFYSVGKLVKVLLVMAVCCLLLCTAPTGADPLPGRDRNTIANKSKDKKASAPKHS LGTGARMALTGGGVLGGVLTNM SEQID52->gi|62546225|gb|AAX86004.1|hyp6.3precursor[Anophelesgambiae] MKFAFAFVLIALFAVFAVSQALPQPEQAAASSNDGASAITKIVLELTPEQAAAVQKM GGRGFWPIMMKSVKKIMAIGCDLIDC SEQID53->gi|17026153|emb|CAD12038.1|Sec61protein[Anophelesgambiae] MGIKFLEIIKPFCGILPEIAKPERKIQFREKVLWTAITLFIFLVCCQIPLFGIMSSDSADPF YWIRVILASNRGTLM SEQID54->gi|62546223|gb|AAX86003.1|hyp6.2precursor[Anophelesgambiae] MGRVMCLLRLMSTLLVVLSIVGKKTNAAPQVTEAPGNVGSTYSPMADIGRLATGAT KLFGQFWNTGTRFGTELSRRTFDFLRVKK SEQID55->gi|18389915|gb|AAL68794.1|AF457564.sub.-1hypotheticalprotein17[Anopheles gambiae] MAGDIQLFSTRETTMKLYSGYRLLVLLVMTVCCLLLFIAPTGADPLPGQTQRTLGYR GNDKRATPPMHSLGSGARMAMTGGGILGGIFSAL SEQID56->gi|87080391|gb|ABD18596.1|defensin[Anophelesgambiae] MDQCSVPRLCIIIMKSFIAAAVIALICAIAVSGTTVTLQSTCKLFTADVVSSITCKMYC VIKGKTGGYCNSEGLCTCRAEDLHFLLKPIINKD SEQID57->gi|18389901|gb|AAL68787.1|AF457557.sub.-1hypotheticalprotein10[Anopheles gambiae] MRFLSVLTVGLLVWVGVFATVNAEDPRTELIGCGSVLFHLAANRLSLQLEEFAVCK RSNPGYDCSDSIHRAISDLQQGLFDLNHCTKDIR SEQID58->gi|18389905|gb|AAL68789.1|AF457559.sub.-1hypotheticalprotein12[Anopheles gambiae] MRFCCVALIGLLLCSVQSVTANDPVDALGACSGNLFGLLMTRLQQMVEDFTACRQE ATANDPQHDRSDSIQRAKVDLQQQLVNYSYCTKNIQ SEQID59->gi|4127344|emb|CAA76832.1|cE5protein[Anophelesgambiae] MASKLFVLAFLCLALVVVVQSAPQYARGDVPTYDEEDFDEESLKPHSSSPSDDGEEE FDPSLLEEHADAPTARDPGRNPEFLRNSNTDEQASAPAASSSDS SEQID60->gi|4210617|emb|CAA10259.1|SG2protein[Anophelesgambiae] MKSMLVAFATLSVALVVVVAIPANFNYGGGGGYFINGTGQSFNFSGESNGTSIPGLP DFGSFLPNLGNLTQQFGGSSGAFPQFSIPSWTNFTDAFTSILPFFGNGQGGGFPFFG SEQID61->gi|4127309|emb|CAA76820.1|hypotheticalprotein[Anophelesgambiae] MTPLIATLAACALTLSIVHSRGLPESSDKLEACGQHYGXLLKASTTWNEKECNGSTK LAACVVSEHEQAYRELKQRCQEAHDERTAKVNAIYEKLPAYLSEVSARVNVLQVSL QHDLPNLQE SEQID62->gi|4375824|emb|CAA76825.1|opsin[Anophelesgambiae] PDVAEPLVHHHLRHLRVLAAAADHHLLVHLHPEGCVRSREEHARAGQEGNVASLR TQEAQNTSTEMKLAKVALVTISLWFMAWTPYLVINFTGIFKAAPISPLATIRGSLFAK ANAVYNPIVYG SEQID63->gi|62546233|gb|AAX86008.1|unknown[Anophelesgambiae] MATTWIPTSVHGPYPPHMVPGGVDSDGAQIFVGRAHHAGDLLPAKVIPDKTAAYVA YGGQETLVEHVEVLVHKQLIWDTASAGQVPLGAVVGGHTSDGEILYVGRAYHEGS QTIGKVQCSHNCIYIPYGGAEVSVPTYEVLCER SEQID64->gi|3378531|emb|CAA03872.1|D7r2protein[Anophelesgambiae] MFKKLLLSVGLVWCLISLGQARKESTVEECEKNIGDSLKDRVCELRQYTPVSSDDM DKHMQCVLEVVGFVDGNGEVKESVLLELLQRVDSGVNHAANMKKCVTEASTSGSD KKANTFYTCFLGTSSLAGFKNAVDYDELLKAGKMQTSDP SEQID65->gi|3378529|emb|CAA03871.1|D7r3protein[Anophelesgambiae] MFGKLLPCAILLWCLFSLGQARQEETVEECERNIPASLKERVCELRQYTPVQGKDMD SHMQCVLEVLGFVEDNGELVFQELLGVLKMVDPDGDHAGSMKKCNGEAEKVDTSS KANTFYTCFLGTSSAQAFKYAVDYVXAXRAGKLDMGTTFNAGQV SEQID66->gi|18389893|gb|AAL68783.1|AF457553.sub.-1mucin-likeprotein[Anopheles gambiae] AGGFSLFEALKQTTTRGEMFRRKLTPTVVVVLLCLTFVADALTIQELRAQIAQQRIQQ RYGVTVATTSAATTTAATTSAATTSEATTTAAASTTQASDSDNTTTTAEATTTTEAQ TTSSSDNSTTTEAAATTTAASETTADSSSTGTTSVEAGLRAQYRDQVRQQAIERALA RAAAFG SEQID67->gi|18389881|gb|AAL68777.1|AF457547.sub.-1selenoprotein[Anophelesgambiae] MRLFAITCLLFSIVTVIGAEFSAEDCRELGLIKSQLFCSACSSLSDYGLIELKEHCLECC QKDTEADSKLKVYPAAVLEVCTCKFGAYPQIQAFIKSDRPAKFPNLTIKYVRGLDPIV KLMDEQGTVKETLSINKWNTDTVQEFFETRLAKVEDDDYIKTNRV SEQID68->gi|18389879|gb|AAL68776.1|AF457546.sub.-130kDaprotein[Anopheles gambiae] MAGAITYICFILHGVSEIIPQQQKKTMKFLLLVASVLCLVLIVSARPADDTSDQESSTE LSDDAGAEEGAEDAGSDAEADAGAADGEEGATDTESGAEGDDSEMDSAMKEGEEG AGSDDAVSGADDETEESKDDAEEDSEEGGEEGGDSASGGEGGEKESPRNTYRQVHK LLKKIMKVDTKD SEQID69->gi|18378603|gb|AAL68639.1|AF458073.sub.-1D7-related5protein[Anopheles gambiae] MEWRYFVVIALICPLIIVETLAVSDCVRHVSESARNTVCDVRQYRVTKGVEADRYVQ CFMTALGFADESGSIQRSNVLTALDAVETHDGVYTDAVDVCLSKAKKLPGTERSGY FFSCMLRTESALNFRDAVELQELRVASKWPEGERFDRSKVQQMMRELNSQLRC SEQID70->gi|18389897|gb|AAL68785.1|AF457555.sub.-1salivarygland1-like4protein [Anophelesgambiae] GREAIETMRTEQRNHRQQLLLLYLDAADLRRALHQYQLLAAQGDRHLPQQIVKFVY AAPRHENRRLENLLDLVRQLPARQDQRTLYQLLQPEIMKRPAQNQSTLAMLTALEM GQVVEGNGELKKQQDAMYQLVLKRWMFLCLAGQYREIVQFATKHPRLFE SEQID71->gi|18389883|gb|AAL68778.1|AF457548.sub.-1antigen5-related1protein [Anophelesgambiae] MAIWIVCATLLLAVLSVVSVGGQYCSSDLCPRGGPHVGCNPPSSSGGPTCQGKQKA RKVLLTPALQAYIMDEHNLNRSNIALGRIRPYPSAVKMPTLTWDPELASLADANARS CNYGHDRCRATKKFPYAGQNIAITQFFGYRFTEKDLIHKFVSSWWSEYLDARPEHVR KYPSSYSG SEQID72->gi|83016748|dbj|BAE53441.1|DsRed[syntheticconstruct] MKLASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVKLKVTKGGPL PFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVATVTQD SSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHKALK LKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH LFLRSRAPPPPPLT SEQID73->gi|18389895|gb|AAL68784.1|AF457554.sub.-1salivarygland1-like3protein [Anophelesgambiae] MAGQRHLIEQAWQYGAQLQHELMLTSMESDRVQRALVLHSMLVNASLAEMVKES YQTHGADGRMVVRMLKFVRLLPGADERVAVYKQLAELLKSNGQDGRFPAVIFSTD VRQLEDRYKPDHAQYEGKVVERWLAELQAGTFHEVVEFARDYPEYFARVEEPLYE TLKQQWSAEGLDRMVSFPNALPVGVQRVRALRALLETLLQHQGEQNNDVYLIRLA HETGRVEATVGQADAAVRQALDDVKKLFEQFKYQRGFPDYEALYKLFKGL SEQID74->gi|18389891|gb|AAL68782.1|AF457552.sub.-1D7proteinlongform[Anopheles gambiae] MIVPRVLLFILLELFVQATQAFKALDPEEAWYVYERCHEDHLPSGPNRETYLKTWKF WKLEPNDAVTHCYVKCTLAGLQMYDEKTNTFKPETVPVQHEAYKSFTEVESSKVN ELQQALSSLNAGSGSCAEVFNAYLPVHNKYIGVSRKIYHGTVDSVAKIYEAKPEIKK QEESFFAYCAKKALGANGKEGYKKIRDYELADSAEFRNAMDCVFRGFRYMDDSGL KVDEVVRDFNLINKSDLEPEVRSVLASCTGTHAYDYYSCLLNSSVKEDFRNAFYFHE LRSANYGYLAMGKVYEGPEKVKEELKKLNY SEQID75->emb|CAC35527.1|gSG9protein[Anophelesgambiae] MCKFYRLISTLLVVVVIAPRHQCSPFFFQYNRPYLSQPSSQLASTAANVVQRSNVTVA LGNRINTDTALDDYGTRV SEQID76->sp|Q9U9L1|RS17.sub.-ANOGA40SribosomalproteinS17 MGRVRTKTIKKASKVIIEKYYTRLTMDFDTNKRIVEEVAIIPTKPLRNKIAGFVTHLM KRLRHSQVRGISIKLQEEERERRDNYVPDVSALEQDIIEVDPETKEMLKHLDFNNIVV QLTNPTAPGYSNRRN SEQID77->emb|CAC35523.1|gSG7protein[Anophelesgambiae] MHAKPAFVLIALGVICLLQTTPTSASTNHVQQLMKVFRSMTQNFDYTKKPSYLQRA KYGVQNQLRNPLVQKAGNLPKSAKLSDGCLKQMVARVTDLEASFYASFSYNCHDH DQYSMECLEAAEPKYLDGLKTLADETAQCMRDQQ SEQID78->gb|AAD47075.1|AF164151.sub.-1translationinitiationfactor4C(1A)[Anopheles gambiae] MPKNKGKGGKNRRRGKNENESEKRELIFKEDEQEYAQVTKMLGNGRLEAMCFDGV KRLCHIRGKLRKKVWINQGDIILIGLRDYQDSKADVILKYTPDEARNLKTYGEFPESV RTNETVTFVENDMDDDIEFGDDYSSSEEGDAIDAI SEQID79->emb|CAC35519.1|gSG2-likeprotein[Anophelesgambiae] MKLFLTLLSTLSVAMVFALPAHHHSRGGDGSSANSTGNSDNNSAGVPDFGFNSQSN VPGFGNGQQPGQQQQGQQGQGFPFFGQGQSGFPSFGNRLQPFFGQNQQGQDGDAQ QGRGVPFFGQGGGQGGIPSFGSGQQNGGVPFLGNGQGQSGFPSFGNGQQGGNFPFFG SEQID80->emb|CAC35451.1|hypotheticalprotein[Anophelesgambiae] MKLYAFALVLCVGLAVGAEVDSVPEVPSDLQQQLDELQLADKPEAPVDDAEQPLPP NGDELPEDAPEPVPEDGSPDEEHLEEEQEEEAEADEEEADESESEESEESDELEEARL VAEELEERQQELDYLKRYLVGRLQAVAILDRRVRPAVIRRPWIRRPWIRRPG SEQID81>emb|CAC35524.1|D7r4protein[Anophelesgambiae] MIRQVIISYFLTVCLLALVQSETVQDCENKLPPSLKSRLCEIRRYEIIEGPEMDKHIHCV MRALDFVYEDGRGDYHKLYDPLNIIELDKRHDVNLEKCIGECVQVPTSERAHVFYK CLLKSTTGRTFKKVFDLMELKKAGKVPQHQRYTAEFVQIMKDYDKALNC SEQID82->ref|XP.sub.-001230998.1|ENSANGP00000014906[Anophelesgambiaestr. PEST] MEAISEALQPYKEQVGMAAGILTVGQMFSGCFVCNDIRKKGTTDGFSAMPFVGGCG LTVLFLQHGMLMNDSAMTNANLVGLTISLAYAIFFLLYTPPTGRSSYWRQVGGTALF TITLLGYVKVENPSVVEDRFGMIITVLMLALIGQPLFGLPDIIRRKSTEGLPFAMILSGT IVGLSWLLYGVILNNVFVVCQNLAAVTLSGIQLALFAIYPSKAAPPSKKRE SEQID83->ref|XP.sub.-316361.2|ENSANGP00000012984[Anophelesgambiaestr.PEST] MESIAVALQPYKDTVGLTAAIVTVVQFFSGVLALNAIRRQGNTRGFSALPFLGGTVF CLLNIQFGQMLRDDGMIRVNFIGLALNLLYVCGFYLYTEGPAKTAVWGQIGLAGAL TAGVLSYVQYEDPQLVEFRFGLILTGLLWTLVGMPLLGLGDILKKKSTEGLPFPIIFLG AVVSFAWLLYGIILRSNFLVVQNLMALALSAVQLSLFIIFPSGAAKPPPTPAKKRN SEQID84->ref|XP.sub.-314140.3|ENSANGP00000015780[Anophelesgambiaestr.PEST] MDGIMSKGSLASLATVATVLQFLTGTVICNRYIRKKSTGDTSAFPFISGFLSCFMWLK YGVLTEESTLILVNFIGSALFFSYTVVFFIFCVNKREVIRQMMVISCIILSATLYTLFETD DEKSIRVIGLLCCCLAVLFFASPLTMLAHVIRTQNTDSLPFPIIMASFFVCLLWTAYGV LIGDRFIQIPNLLGGILAGIQLTLYVIYPKKKASFSGGPRYSPLVSENPIL SEQID85->emb|CAC35522.1|gSG6protein[Anophelesgambiae] MAIRVELLLAMVLLPLLLLESVVPYAAAEKVWVDRDKVYCGHLDCTRVATFKGER FCTLCDTRHFCECKETREPLPYMYACPGTEPCQSSDRLGSCSKSMHDVLCDRIDQAF LEQ

(16) The peptides of the present invention, such as those of SEQ ID 1-44 and those within SEQ ID 131-134 described above preferably comprise one or more further amino acids at one or both of their termini in order to aid in their processing into vaccines. Typically, these further amino acids are the ones adjacent to each of the termini of SEQ ID 1-44, as shown in the larger proteins of SEQ ID 45-85 above (these larger proteins contain the sequences of SEQ ID 1-44). Preferably the number of further amino acids at each terminus is from 1-5, more preferably from 1-3, and most preferably 2 at each terminus. In each of these cases, if there are less than two further amino acids at that terminus of the sequences of SEQ ID 1-44, then the further amino acids include all of the remaining amino acid(s) at that terminus Particularly preferred sequences of this type, corresponding to SEQ ID 1-44, are as follows:

(17) TABLE-US-00010 SEQID86 MHLTLFTVAVLLLAAAALLLLLPPAYSTTLTPPAP SEQID87 MPLSYCHLFLTHTLARALSFSRSDCLKF SEQID88 MKNVFFALLLVVLVCCLVSVQGNEIIQ SEQID89 GIKLLVLLICLFFYHTHCTTAYLWLAMGVEA SEQID90 MFLKGSFPRFQMCVMLIGFFSSAKCLMC SEQID91 KQNDYQALLGLCCPWIDLAAADLPMRRHAKARE SEQID92 MKFYSVGKLVKVLLVMAVCCLLLCTAPTGADPLPG SEQID93 MKFAFAFVLIALFAVFAVSQALPQPEQAAASS SEQID94 SNDGASAITKIVLELTPEQAAAVQK SEQID95 AITLFIFLVCCQIPLFGIMSSDSADPFYWIRVILASN SEQID96 MGRVMCLLRLMSTLLVVLSIVGKKT SEQID97 MKLYSGYRLLVLLVMTVCCLLLFIAPTGADPLPGQTQRTLGY SEQID98 CKMYCVIKGKTGGYCNSEGLCTCRAEDLHFLLKPIINKD SEQID99 TVNAEDPRTELIGCGSVLFHLAANRLSLQLEEFAVCKRSN SEQID100 CVALIGLLLCSVQSVTANDPVDALGACSGNLFGLLMTRLQQ SEQID101 MASKLFVLAFLCLALVVVVQSAPQYARGDVPTYD SEQID102 MKSMLVAFATLSVALVVVVAIPANFNYGGGGGYFINGTGQSF SEQID103 NAIYEKLPAYLSEVSARVNVLQVSLQHDLPNLQE SEQID104 STEMKLAKVALVTISLWFMAWTPYLVINFTGIFK SEQID105 GDLLPAKVIPDKTAAYVAYGGQETLVEHVEVLVHK SEQID106 NTFYTCFLGTSSLAGFKNAVDYDELLKAGKM SEQID107 QCVLEVLGFVEDNGELVFQELLGVLKMVDPDGDHA SEQID108 RRKLTPTVVVVLLCLTFVADALTIQELRAQIAQQRIQQRYGVTVATTSA SEQID109 CSSLSDYGLIELKEHCLECCQKDTEADSKLKVYPAAVLEVCT SEQID110 AITYICFILHGVSEIIPQQQKKTMKFLLLVASVLCLVLIVS SEQID111 EWRYFVVIALICPLIIVETLAVSD SEQID112 QLLLLYLDAADLRRALHQYQLLAAQGDRHLPQQIVKFVYA SEQID113 RKVLLTPALQAYIMDEHNLNRSNIALGRIRPYPSAVKMPTL SEQID114 DGVLKGETHKALKLKDGGHYLVEFKSIYMAK SEQID115 ALVLHSMLVNASLAEMVKESYQTHGADGRMVVRMLKFVRLLPGA SEQID116 VQRVRALRALLETLLQHQGEQNNDVYLIRLAHETGR SEQID117 VNELQQALSSLNAGSGSCAEVFNAYLPVHNKYIGVSRKIYH SEQID118 MCKFYRLISTLLVVVVIAPRHQCSPFFFQYNRPYLSQ SEQID119 RDNYVPDVSALEQDIIEVDPETKEMLKHLDFNNIVVQLTN SEQID120 HDQYSMECLEAAEPKYLDGLKTLADETAQCMR SEQID121 EQEYAQVTKMLGNGRLEAMCFDGVKRLCHIRGKLRK SEQID122 MKLFLTLLSTLSVAMVFALPAHHHSRGGD SEQID123 SDELEEARLVAEELEERQQELDYLKRYLVGRLQAVAI SEQID124 IISYFLTVCLLALVQSETVQDCE SEQID125 DSAMTNANLVGLTISLAYAIFFLLYTPPTGRSSYW SEQID126 VVSFAWLLYGIILRSNFLVVQNLMALALSAVQLSLFIIFP SEQID127 TSAFPFISGFLSCFMWLKYGVLTEESTLILVNFIGSALFF SEQID128 VIGLLCCCLAVLFFASPLTMLAHVIRTQ SEQID129 VELLLAMVLLPLLLLESVVPYAAAEKVWVD

(18) Particularly preferred such peptides also include the following: residues 1-35 of >gi|18389913|gb|AAL68793.1|AF457563_1 hypothetical protein 16 [Anopheles gambiae] residues 1-28 of >gi|18389909|gb|AAL68791.1|AF457561_1 hypothetical protein 14 [Anopheles gambiae] residues 1-27 of >gi|18389907|gb|AAL68790.1|AF457560_1 hypothetical protein 13 [Anopheles gambiae] residues 8-38 of >gi|18389903|gb|AAL68788.1|AF457558_1 hypothetical protein 11 [Anopheles gambiae] residues 1-28 of >gi|62546227|gb|AAX86005.1|hyp3.5 precursor [Anopheles gambiae] residues 12-44 of gi|18389899|gb|AAL68786.1|AF457556_1 salivary gland 7-like protein [Anopheles gambiae] residues 1-35 of >gi|18389911|gb|AAL68792.1|AF457562_1 hypothetical protein 15 [Anopheles gambiae] residues 1-32 of >gi|62546225|gb|AAX86004.1|hyp6.3 precursor [Anopheles gambiae] residues 32-56 of >gi|62546225|gb|AAX86004.1|hyp6.3 precursor [Anopheles gambiae] residues 36-72 of >gi|17026153|emb|CAD12038.1|Sec61 protein [Anopheles gambiae] residues 1-25 of >gi|62546223|gb|AAX86003.1|hyp6.2 precursor [Anopheles gambiae] residues 15-56 of >gi|18389915|gb|AAL68794.1|AF457564_1 hypothetical protein 17 [Anopheles gambiae] residues 55-93 of >gi|87080391|gb|ABD18596.1|defensin [Anopheles gambiae] residues 20-59 of >gi|18389901|gb|AAL68787.1|AF457557_1 hypothetical protein 10 [Anopheles gambiae] residues 5-45 of >gi|18389905|gb|AAL68789.1|AF457559_1 hypothetical protein 12 [Anopheles gambiae] residues 1-34 of >gi|4127344|emb|CAA76832.1| cE5 protein [Anopheles gambiae] residues 1-42 of >gi|4210617|emb|CAA10259.1| SG2 protein [Anopheles gambiae] residues 89-122 of >gi|4127309|emb|CAA76820.1| hypothetical protein [Anopheles gambiae] residues 63-96 of >gi|4375824|emb|CAA76825.1| opsin [Anopheles gambiae] residues 39-73 of >gi|62546233|gb|AAX86008.1| unknown [Anopheles gambiae] residues 115-145 of >gi|3378531|emb|CAA03872.1|D7r2 protein [Anopheles gambiae] residues 61-95 of >gi|3378529|emb|CAA03871.1|D7r3 protein [Anopheles gambiae] residues 21-69 of >gi|18389893|gb|AAL68783.1|AF457553_1 mucin-like protein [Anopheles gambiae] residues 41-82 of >gi|18389881|gb|AAL68777.1|AF457547_1 selenoprotein [Anopheles gambiae] residues 4-44 of >gi|18389879|gb|AAL68776.1|AF457546_1 30 kDa protein [Anopheles gambiae] residues 2-25 of >gi|18378603|gb|AAL68639.1|AF458073_1 D7-related 5 protein [Anopheles gambiae] residues 18-57 of >gi|18389897|gb|AAL68785.1|AF457555_1 salivary gland 1-like 4 protein [Anopheles gambiae] residues 57-97 of >gi|18389883|gb|AAL68778.1|AF457548_1 antigen 5-related 1 protein [Anopheles gambiae] residues 156-186 of >gi|83016748|dbj|BAE53441.1|DsRed [synthetic construct] residues 35-78 of >gi|18389895|gb|AAL68784.1|AF457554_1 salivary gland 1-like 3 protein [Anopheles gambiae] residues 189-224 of >gi|18389895|gb|AAL68784.1|AF457554_1 salivary gland 1-like 3 protein [Anopheles gambiae] residues 111-151 of >gi|18389891|gb|AAL68782.1|AF457552_1 D7 protein long form [Anopheles gambiae] residues 1-37 of >emb|CAC35527.1| gSG9 protein [Anopheles gambiae] residues 81-120 of >sp|Q9U9L1|RS17_ANOGA 40S ribosomal protein S17 residues 111 to 142 of >emb|CAC35523.1| gSG7 protein [Anopheles gambiae] residues 32-67 of >gb|AAD47075.1|AF164151_1 translation initiation factor 4C (1A) [Anopheles gambiae] residues 1-29 of >emb|CAC35519.1| gSG2-like protein [Anopheles gambiae] residues 106-142 of >emb|CAC35451.1|hypothetical protein [Anopheles gambiae] residues 6-28 of >emb|CAC35524.1| D7r4 protein [Anopheles gambiae] residues 70-104 of >ref|XP_001230998.1|ENSANGP00000014906 [Anopheles gambiae str. PEST] residues 174-213 of >ref|XP_316361.2|ENSANGP00000012984 [Anopheles gambiae str. PEST] residues 41-80 of >ref|XP_314140.3|ENSANGP00000015780 [Anopheles gambiae str. PEST] residues 126-153 of >ref|XP_314140.3|ENSANGP00000015780 [Anopheles gambiae str. PEST] residues 5-34 of >emb|CAC35522.1| gSG6 protein [Anopheles gambiae]

(19) In alternative embodiments of the present invention, the invention is directed to compositions comprising polypeptides which are homologous to those described above, in particular peptides that are homologous to any of SEQ ID 1-134. The homology referred to above in respect of these sequences is preferably 60%, 75%, 80%, 85%, 90%, 95% or substantially 100%.

(20) The percent homology of a first polypeptide sequence to a second polypeptide sequence, as referred to in the context of the present invention, is defined as the number of amino acid residues in the second sequence that match in both position and identity to those in the first sequence, divided by the total number of amino acid residues in the second polypeptide (both first and second polypeptides must have the same number of amino acid residues) and multiplied by 100. In the present invention, it is preferred that the polypeptide homology to the defined sequences is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% (or substantially 100%).

(21) In the present invention, the arthropod borne disease is not especially limited, and the polypeptides may be immunogenic against, and/or derived from, any known arthropod borne disease. Examples of diseases, pathogens and vectors covered by the present invention, are set out in Table 1 above. Preferably, however, the relevant disease is malaria (including any malaria strain), as caused by any one of the strains of Plasmodium.

(22) The specific sequences homologous to any of SEQ ID 1-134 described above are preferably the ones at the appropriate positions within known arthropod proteins, which can be found at the public NCBI protein database, which may be accessed online at the following URL address http://www.ncbi.nlm.nih.gov/entrez/query/static/help/helpdoc.html#Protein. The list is typically in the form |version number (gi number)|database identification (e.g. gb for GenBank)|NCBI accession number|optional further information (e.g. the accession number of the nucleotide sequence from which the protein sequence is derived). The sequences The protein database contains sequence data from the translated coding regions from DNA sequences in GenBank, EMBL, and DDBJ as well as protein sequences submitted to Protein Information Resource (PIR), SWISS-PROT, Protein Research Foundation (PRF), and Protein Data Bank (PDB) (sequences from solved structures).

(23) The epitopes within the sequences defined above are not especially limited, provided that they contain 7 amino acid residues or more. Preferably the epitopes are at least of a length that is appropriate for the smaller immunogenic epitopes, such as CTL, T helper and B cell epitopes in a particular vertebrate species, such as in a human. Typically the epitopes contain 8, 9, 10, or 11 amino acid residues, but may contain more if desired.

(24) Although it may comprise more amino acids typically, the polypeptide comprises 100 amino acids or less, preferably between 7 and 100 amino acids, and more preferably from 8-75 amino acids. The size should not be so great that useful epitopes suffer from competition with non-protective epitopes in the immune system (for this reason full proteins are not included), nor should the size be so small that only a very narrow range of protection is offered. More preferred ranges are from 15-75 amino acids, 20-55 amino acids and 23-50 amino acids. It is particularly preferred that the polypeptide consists of (or substantially consists of) a sequence selected from the sequences defined above.

(25) In addition to the polypeptides described above, the invention also provides multi-epitope immunogenic polypeptides comprising two or more polypeptides of the present invention either as multi-branched polypeptides or concatenated sequences. These multi-epitope polypeptides are not limited in size and may comprise e.g. up to 1400, or up to 900, or up to 550 amino acids. Thus, they extend not only to the polypeptides outlined above, but also to larger polypeptides, provided that these larger polypeptides comprise two or more units, each unit consisting of a polypeptide of the invention. Thus, a polypeptide having 100 repeating units of a 7-mer according to the present invention is encompassed by the present invention, as is a polypeptide having, say 52 units of one 8-mer epitope, and 23 units of a second 10-mer epitope. Polypeptides of this type will not suffer from the competition problems associated with similar length polypeptides that comprise only one or two epitopes. For the avoidance of doubt, the multi-epitope polypeptide may comprise multiple copies of the same epitope, or single copies of a plurality of different epitopes, or multiple copies of 2 or more epitopes. It is particularly preferred that a multi-epitope polypeptide comprises two or more of the sequences described above in SEQ ID 1-44 (and especially those in SEQ ID 1-6, 7, 8, 11, 12, 15, 16, 20, 26, 28, 30-32, 35, 41, and 42) or in SEQ ID 86-134.

(26) As has been mentioned, the invention provides a polypeptide composition comprising one or more, preferably two or more different polypeptides as defined above. Thus, the polypeptide composition may comprise any number of polypeptides of the present invention together in the same sequence, mixture or formulation. The presence of a plurality of polypeptides together is useful since each may elicit its own immune response, widening the protective effect of the composition. It is particularly preferred that the composition contains two or more of (or all of) the sequences of SEQ ID 1-44 (and especially those in SEQ ID 1-6, 7, 8, 11, 12, 15, 16, 20, 26, 28, 30-32, 35, 41, and 42), and/or two or more of the epitopes within SEQ ID 86-134. In the composition each sequence and/or epitope may be present either as a separate peptide, or as a number of larger peptides comprising several concatenated epitopes and/or sequences (e.g. three sequences concatenated in one larger peptide and another 4 in another larger peptide, etc.).

(27) The invention also provides a polypeptide construct, which construct comprises a polypeptide as defined above and a carrier. The construct may be formed by combining one or more epitopes and/or polypeptides as defined above with the carrier. The carrier may be a molecule, such as an adjuvant and/or an excipient. Combining in this context means either mixing together, or attaching together (e.g. via a covalent linkage).

(28) The present invention further provides a polypeptide as defined above for use in medicine. Also provided is a medicament or vaccine composition against arthropod borne diseases, comprising a polypeptide as defined above, and one or more appropriate excipients and/or adjuvants, or a polypeptide construct as defined above and optionally one or more appropriate excipients and/or adjuvants (if the carrier part of the construct is itself an excipient or adjuvant, then a further excipient or adjuvant may not be needed). The excipient or adjuvant is not especially limited, and any excipients or adjuvants used in medicaments and vaccines may be employed. The medicament or vaccine composition may be produced according to any known method appropriately adapted to the present invention, such as by mixing a polypeptide of the invention with an appropriate excipient.

(29) A method of producing a polypeptide as defined above is also provided by the invention. The method is not especially limited, and typically comprises joining two or more epitopes to form the polypeptide. The polypeptide may, however, be synthesised by direct chemical synthesis (e.g. incorporating one amino acid at a time until the full polypeptide is formed) or by recombinant methods. Such general methods are well known to the skilled person and may be adapted to the present invention as desired. In some instances, the polypeptide of the present invention may comprise additional amino acid sequences at one or both termini to help in synthesis of the polypeptide. These additional sequences are preferably from 1-5 amino acids in length. Typically 2 amino acids are involved. Examples of such sequences are provided as SEQ ID 86-129, as described above.

(30) The invention still further provides use of a polypeptide or composition as defined above, in the manufacture of a medicament or vaccine, effective in the treatment or prevention of an arthropod borne disease. Also provided is a method of treating or preventing an arthropod borne disease, which method comprises administering a polypeptide, a composition, a medicament or a vaccine as defined above to a vertebrate. The method of administration is not especially limited, and may comprise subcutaneous, intramuscular, intravenous, intra-dermal, or intra-nasal administration, or may be administered orally (e.g. in the form of a pill or a liquid preparation), or may be in the form of a suppository, if desired. The form of such administration preparations is not especially limited, and known forms may be employed with appropriate modifications that will be apparent to the skilled person. The dosage is not especially limited and may range from 1 pg to 100 g, preferably 1 ng to 100 g of the polypeptide per individual, depending upon the size, weight and species of the individual involved.

(31) The invention may be applied to any vertebrate, since the immune systems of vertebrates operate in a related manner. Typically, the vertebrate referred to in the present context is a mammal, bird, a reptile or a fish. It is especially preferred that the vertebrate is a human, a domestic animal (such as a dog or a cat), a farm animal (such as a pig or a horse), a bovine animal (such as cattle), or fowl (such as a domestic bird, a farm bird, or a game bird). When the vertebrate is a bird, it is preferably a chicken, a turkey, a duck, or a goose.

(32) Examples of human MHCs (HLAs) that may be associated with a particular T cell epitope in the present invention include the following:

(33) HLA-A

(34) A*010101, A*010102, A*010103, A*0102, A*0103, A*0104N, A*0106, A*0107, A*0108, A*0109, A*0110, A*02010101, A*02010102L, A*020102, A*020103, A*020104, A*020105, A*020106, A*020107, A*020108, A*020109, A*020110, A*020111, A*0202, A*020301, A*020302, A*0204, A*0205, A*020601, A*020602, A*020603, A*0207, A*0208, A*0209, A*0210, A*0211, A*0212, A*0213, A*0214, A*0215N, A*0216, A*021701, A*021702, A*0218, A*0219, A*022001, A*022002, A*0221, A*0222, A*0224, A*0225, A*0226, A*0227, A*0228, A*0229, A*0230, A*0231, A*0232N, A*0233, A*0234, A*023501, A*023502, A*0236, A*0237, A*0238, A*0239, A*0240, A*0241, A*0242, A*0243N, A*0244, A*0245, A*0246, A*0247, A*0248, A*0249, A*0250, A*0251, A*0252, A*0253N, A*0254, A*0255, A*0256, A*0257, A*0258, A*0259, A*0260, A*0261, A*0262, A*0263, A*0264, A*0265, A*0266, A*0267, A*0268, A*0269, A*0270, A*0271, A*0272, A*0273, A*03010101, A*03010102N, A*03010103, A*030102, A*030103, A*0302, A*0303N, A*0304, A*0305, A*0306, A*0307, A*0308, A*0309, A*0310, A*0311N, A*0312, A*0313, A*0314, A*110101, A*110102, A*1102, A*1103, A*1104, A*1105, A*1106, A*1107, A*1108, A*1109, A*1110, A*1111, A*1112, A*1113, A*1114, A*1115, A*1116, A*1117, A*1118, A*1119, A*2301, A*2302, A*2303, A*2304, A*2305, A*2306, A*2307N, A*2308N, A*2309, A*2310, A*2311N, A*2312, A*24020101, A*24020102L, A*240202, A*240203, A*240204, A*240205, A*240206, A*240301, A*240302, A*2404, A*2405, A*2406, A*2407, A*2408, A*2409N, A*2410, A*2411N, A*2413, A*2414, A*2415, A*2417, A*2418, A*2419, A*2420, A*2421, A*2422, A*2423, A*2424, A*2425, A*2426, A*2427, A*2428, A*2429, A*2430, A*2431, A*2432, A*2433, A*2434, A*2435, A*2436N, A*2437, A*2438, A*2439, A*2440N, A*2441, A*2442, A*2443, A*2444, A*2445N, A*2446, A*250101, A*250102, A*2502, A*2503, A*2504, A*2601, A*2602, A*2603, A*2604, A*2605, A*2606, A*260701, A*260702, A*2608, A*2609, A*2610, A*2611N, A*2612, A*2613, A*2614, A*2615, A*2616, A*2617, A*2618, A*2619, A*2620, A*2621, A*2622, A*2623, A*29010101, A*29010102N, A*290201, A*290202, A*290203, A*2903, A*2904, A*2905, A*2906, A*2907, A*2908N, A*2909, A*2910, A*2911, A*300101, A*300102, A*300201, A*300202, A*3003, A*3004, A*3006, A*3007, A*3008, A*3009, A*3010, A*3011, A*3012, A*310102, A*3102, A*3103, A*3104, A*3105, A*3106, A*3107, A*3108, A*3109, A*3110, A*3201, A*3202, A*3203, A*3204, A*3205, A*3206, A*3207, A*3208, A*3301, A*330301, A*330302, A*3304, A*3305, A*3306, A*3307, A*3401, A*3402, A*3403, A*3404, A*3405, A*3406, A*3601, A*3602, A*3603, A*3604, A*4301, A*6601, A*6602, A*6603, A*6604, A*680101, A*680102, A*680103, A*6802, A*680301, A*680302, A*6804, A*6805, A*6806, A*6807, A*6808, A*6809, A*6810, A*6811N, A*6812, A*6813, A*6814, A*6815, A*6816, A*6817, A*6818N, A*6819, A*6820, A*6821, A*6822, A*6823, A*6824, A*6825, A*6826, A*6827, A*6901, A*7401, A*7402, A*7403, A*7404, A*7405, A*7406, A*7407, A*7408, A*7409, A*7410, A*8001.

(35) HLA-B

(36) B*070201, B*070202, B*070203, B*070204, B*0703, B*0704, B*0705, B*0706, B*0707, B*0708, B*0709, B*0710, B*0711, B*0712, B*0713, B*0714, B*0715, B*0716, B*0717, B*0718, B*0719, B*0720, B*0721, B*0722, B*0723, B*0724, B*0725, B*0726, B*0727, B*0728, B*0729, B*0730, B*0731, B*0732, B*0733, B*0734, B*0735, B*0736, B*0737, B*0738, B*0801, B*0802, B*0803, B*0804, B*0805, B*0806, B*0807, B*0808N, B*0809, B*0810, B*0811, B*0812, B*0813, B*0814, B*0815, B*0816, B*0817, B*0818, B*0819N, B*0820, B*0821, B*0822, B*1301, B*1302, B*1303, B*1304, B*1306, B*1307N, B*1308, B*1309, B*1310, B*1311, B*1312, B*1313, B*1401, B*1402, B*1403, B*1404, B*1405, B*140601, B*140602, B*15010101, B*15010102N, B*150102, B*150103, B*150104, B*150105, B*1502, B*1503, B*1504, B*1505, B*1506, B*1507, B*1508, B*1509, B*1510, B*151101, B*151102, B*1512, B*1513, B*1514, B*1515, B*1516, B*15170101, B*15170102, B*1518, B*1519, B*1520, B*1521, B*1523, B*1524, B*1525, B*1526N, B*1527, B*1528, B*1529, B*1530, B*1531, B*1532, B*1533, B*1534, B*1535, B*1536, B*1537, B*1538, B*1539, B*1540, B*1542, B*1543, B*1544, B*1545, B*1546, B*1547, B*1548, B*1549, B*1550, B*1551, B*1552, B*1553, B*1554, B*1555, B*1556, B*1557, B*1558, B*1560, B*1561, B*1562, B*1563, B*1564, B*1565, B*1566, B*1567, B*1568, B*1569, B*1570, B*1571, B*1572, B*1573, B*1574, B*1575, B*1576, B*1577, B*1578, B*1579N, B*1580, B*1581, B*1582, B*1583, B*1584, B*1585, B*1586, B*1587, B*1588, B*1589, B*1590, B*1591, B*1592, B*1593, B*1594N, B*180101, B*180102, B*1802, B*1803, B*1804, B*1805, B*1806, B*1807, B*1808, B*1809, B*1810, B*1811, B*1812, B*1813, B*1814, B*1815, B*1817N, B*1818, B*1819, B*1820, B*2701, B*2702, B*2703, B*2704, B*270502, B*270503, B*270504, B*270505, B*270506, B*270507, B*2706, B*2707, B*2708, B*2709, B*2710, B*2711, B*2712, B*2713, B*2714, B*2715, B*2716, B*2717, B*2718, B*2719, B*2720, B*2721, B*2723, B*2724, B*2725, B*2726, B*350101B*350102, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*350901, B*350902, B*3510, B*3511, B*3512, B*3513, B*351401, B*351402, B*3515, B*3516, B*3517, B*3518, B*3519, B*3520, B*3521, B*3522, B*3523, B*3524, B*3525, B*3526, B*3527, B*3528, B*3529, B*3530, B*3531, B*3532, B*3533, B*3534, B*3535, B*3536, B*3537, B*3538, B*3539, B*3540N, B*3541, B*3542, B*3543, B*3544, B*3545, B*3546, B*3547, B*3548, B*3549, B*3550, B*3551, B*3552, B*3553N, B*3701, B*3702, B*3703N, B*3704, B*3705, B*3706, B*3707, B*3801, B*380201, B*380202, B*3803, B*3804, B*3805, B*3806, B*3807, B*3808, B*3809, B*3810, B*390101, B*390103, B*390104, B*390201, B*390202, B*3903, B*3904, B*3905, B*390601, B*390602, B*3907, B*3908, B*3909, B*3910, B*3911, B*3912, B*3913, B*3914, B*3915, B*3916, B*3917, B*3918, B*3919, B*3920, B*3922, B*3923, B*3924, B*3925N, B*3926, B*3927, B*3928, B*3929, B*3930, B*3931, B*3932, B*400101, B*400102, B*400103, B*400104, B*400105, B*400201, B*400202, B*4003, B*4004, B*4005, B*40060101, B*40060102, B*4007, B*4008, B*4009, B*4010, B*4011, B*4012, B*4013, B*401401, B*401402, B*401403, B*4015, B*4016, B*4018, B*4019, B*4020, B*4021, B*4022N, B*4023, B*4024, B*4025, B*4026, B*4027, B*4028, B*4029, B*4030, B*4031, B*4032, B*4033, B*4034, B*4035, B*4036, B*4037, B*4038, B*4039, B*4040, B*4042, B*4043, B*4044, B*4045, B*4046, B*4047, B*4048, B*4049, B*4050, B*4051, B*4052, B*4053, B*4054, B*4055, B*4056, B*4057, B*4101, B*4102, B*4103, B*4104, B*4105, B*4106, B*4201, B*4202, B*4204, B*420501, B*420502, B*4206, B*44020101, B*44020102S, B*440202, B*440203, B*440301, B*440302, B*4404, B*4405, B*4406, B*4407, B*4408, B*4409, B*4410, B*4411, B*4412, B*4413, B*4414, B*4415, B*4416, B*4417, B*4418, B*4419N, B*4420, B*4421, B*4422, B*4423N, B*4424, B*4425, B*4426, B*4427, B*4428, B*4429, B*4430, B*4431, B*4432, B*4433, B*4434, B*4435, B*4436, B*4437, B*4438, B*4439, B*4440, B*4501, B*4502, B*4503, B*4504, B*4505, B*4506, B*4507, B*4601, B*4602, B*4603, B*4604, B*47010101, B*47010102, B*4702, B*4703, B*4704, B*4705, B*4801, B*4802, B*4803, B*4804, B*4805, B*4806, B*4807, B*4808, B*4809, B*4810, B*4901, B*4902, B*4903, B*5001, B*5002, B*5004, B*510101, B*510102, B*510103, B*510104, B*510105, B*510201, B*510202, B*5103, B*5104, B*5105, B*5106, B*5107, B*5108, B*5109, B*5110, B*5111N, B*5112, B*511301, B*511302, B*5114, B*5115, B*5116, B*5117, B*5118, B*5119, B*5120, B*5121, B*5122, B*5123, B*5124, B*5126, B*5127N, B*5128, B*5129, B*5130, B*5131, B*5132, B*5133, B*5134, B*5135, B*5136, B*520101, B*520102, B*520103, B*520104, B*5202, B*5203, B*5204, B*5205, B*5206, B*530101, B*530102, B*5302, B*5303, B*5304, B*5305, B*5306, B*5307, B*5308, B*5309, B*5401, B*5402, B*5501, B*5502, B*5503, B*5504, B*5505, B*5507, B*5508, B*5509, B*5510, B*5511, B*5512, B*5513, B*5514, B*5515, B*5516, B*5601, B*5602, B*5603, B*5604, B*560501, B*560502, B*5606, B*5607, B*5608, B*5609, B*5610, B*5611, B*5612, B*5613, B*5614, B*570101, B*570102, B*5702, B*570301, B*570302, B*5704, B*5705, B*5706, B*5707, B*5708, B*5709, B*5801, B*5802, B*5804, B*5805, B*5806, B*5807, B*5808, B*5809, B*5810N, B*5901, B*670101, B*670102, B*6702, B*7301, B*7801, B*780201, B*780202, B*7803, B*7804, B*7805, B*8101, B*8102, B*8201, B*8202, B*8301.

(37) HLA-C

(38) Cw*010201, Cw*010202, Cw*0103, Cw*0104, Cw*0105, Cw*0106, Cw*0107, Cw*0108, Cw*0109, Cw*0110, Cw*020201, Cw*020202, Cw*020203, Cw*020204, Cw*020205, Cw*0203, Cw*0204, Cw*0205, Cw*0206, Cw*0207, Cw*0208, Cw*0209, Cw*030201, Cw*030202, Cw*030301, Cw*030302, Cw*030303, Cw*030304, Cw*030401, Cw*030402, Cw*030403, Cw*0305, Cw*0306, Cw*0307, Cw*0308, Cw*0309, Cw*0310, Cw*0311, Cw*0312, Cw*0313, Cw*0314, Cw*0315, Cw*0316, Cw*0317, Cw*0318, Cw*04010101, Cw*04010102, Cw*040102, Cw*0403, Cw*040401, Cw*040402, Cw*0405, Cw*0406, Cw*0407, Cw*0408, Cw*0409N, Cw*0410, Cw*0411, Cw*0412, Cw*0413, Cw*0414, Cw*0415, Cw*050101, Cw*050102, Cw*0502, Cw*0503, Cw*0504, Cw*0505, Cw*0506, Cw*0507N, Cw*0508, Cw*0509, Cw*0510, Cw*0602, Cw*0603, Cw*0604, Cw*0605, Cw*0606, Cw*0607, Cw*0608, Cw*0609, Cw*0610, Cw*0611, Cw*070101, Cw*070102, Cw*070103, Cw*07020101, Cw*07020102, Cw*07020103, Cw*0703, Cw*070401, Cw*070402, Cw*0705, Cw*0706, Cw*0707, Cw*0708, Cw*0709, Cw*0710, Cw*0711, Cw*0712, Cw*0713, Cw*0714, Cw*0715, Cw*0716, Cw*0717, Cw*0718, Cw*0719, Cw*0720, Cw*0721, Cw*0722, Cw*0723, Cw*0724, Cw*0725, Cw*0726, Cw*0727, Cw*0728, Cw*0729, Cw*080101, Cw*080102, Cw*0802, Cw*0803, Cw*0804, Cw*0805, Cw*0806, Cw*0807, Cw*0808, Cw*0809, Cw*0810, Cw*0811, Cw*0812, Cw*120201, Cw*120202, Cw*120203, Cw*120301, Cw*120302, Cw*120303, Cw*120401, Cw*120402, Cw*1205, Cw*1206, Cw*1207, Cw*1208, Cw*1209, Cw*1210, Cw*1211, Cw*1212, Cw*1213, Cw*1214, Cw*1215, Cw*140201, Cw*140202, Cw*140203, Cw*1403, Cw*1404, Cw*1405, Cw*150201, Cw*150202, Cw*1503, Cw*1504, Cw*150501, Cw*150502, Cw*150503, Cw*150504, Cw*1506, Cw*1507, Cw*1508, Cw*1509, Cw*1510, Cw*1511, Cw*1512, Cw*1601, Cw*1602, Cw*160401, Cw*1606, Cw*1701, Cw*1702, Cw*1703, Cw*1801, Cw*1802.

(39) HLA-E

(40) E*0101, E*010301, E*010302, E*010303, E*0104.

(41) HLA-F

(42) F*010101, F*010102.

(43) HLA-G

(44) G*010101, G*010102, G*010103, G*010104, G*010105, G*010106, G*010107, G*010108, G*0102, G*0103, G*010401, G*010402, G*010403, G*0105N, G*0106.

(45) HLA-DRA

(46) DRA*0101, DRA*010201, DRA*010202.

(47) HLA-DRB1

(48) DRB1*010101, DRB1*010102, DRB1*010103, DRB1*010201, DRB1*010202, DRB1*010203, DRB1*010204, DRB1*0103, DRB1*0104, DRB1*0105, DRB1*0106, DRB1*0107, DRB1*0108, DRB1*0109, DRB1*0110, DRB1*0111, DRB1*030101, DRB1*030102, DRB1*030201, DRB1*030202, DRB1*0303, DRB1*0304, DRB1*030501, DRB1*030502, DRB1*0306, DRB1*0307, DRB1*0308, DRB1*0309, DRB1*0310, DRB1*0311, DRB1*0312, DRB1*0313, DRB1*0314, DRB1*0315, DRB1*0316, DRB1*0317, DRB1*0318, DRB1*0319, DRB1*0320, DRB1*0321, DRB1*0322, DRB1*0323, DRB1*0324, DRB1*0325, DRB1*0326, DRB1*0327, DRB1*0328, DRB1*040101, DRB1*040102, DRB1*0402, DRB1*040301, DRB1*040302, DRB1*0404, DRB1*040501, DRB1*040502, DRB1*040503, DRB1*040504, DRB1*0406, DRB1*040701, DRB1*040702, DRB1*040703, DRB1*0408, DRB1*0409, DRB1*0410, DRB1*0411, DRB1*0412, DRB1*0413, DRB1*0414, DRB1*0415, DRB1*0416, DRB1*0417, DRB1*0418, DRB1*0419, DRB1*0420, DRB1*0421, DRB1*0422, DRB1*0423, DRB1*0424, DRB1*0425, DRB1*0426, DRB1*0427, DRB1*0428, DRB1*0429, DRB1*0430, DRB1*0431, DRB1*0432, DRB1*0433, DRB1*0434, DRB1*0435, DRB1*0436, DRB1*0437, DRB1*0438, DRB1*0439, DRB1*0440, DRB1*0441, DRB1*0442, DRB1*0443, DRB1*0444, DRB1*0445, DRB1*0446, DRB1*0447, DRB1*0448, DRB1*0449, DRB1*0450, DRB1*070101, DRB1*070102, DRB1*0703, DRB1*0704, DRB1*0705, DRB1*0706, DRB1*0707, DRB1*0708, DRB1*080101, DRB1*080102, DRB1*080201, DRB1*080202, DRB1*080203, DRB1*080302, DRB1*080401, DRB1*080402, DRB1*080403, DRB1*080404, DRB1*0805, DRB1*0806, DRB1*0807, DRB1*0808, DRB1*0809, DRB1*0810, DRB1*0811, DRB1*0812, DRB1*0813, DRB1*0814, DRB1*0815, DRB1*0816, DRB1*0817, DRB1*0818, DRB1*0819, DRB1*0820, DRB1*0821, DRB1*0822, DRB1*0823, DRB1*0824, DRB1*0825, DRB1*0826, DRB1*0827, DRB1*0828, DRB1*0829, DRB1*090102, DRB1*090103, DRB1*0902, DRB1*0903, DRB1*100101, DRB1*100102, DRB1*110101, DRB1*110102, DRB1*110103, DRB1*110104, DRB1*110105, DRB1*1102, DRB1*1103, DRB1*110401, DRB1*110402, DRB1*1105, DRB1*110601, DRB1*110602, DRB1*1107, DRB1*110801, DRB1*110802, DRB1*1109, DRB1*1110, DRB1*1111, DRB1*111201, DRB1*111202, DRB1*1113, DRB1*1114, DRB1*1115, DRB1*1116, DRB1*1117, DRB1*1118, DRB1*1119, DRB1*1120, DRB1*1121, DRB1*1122, DRB1*1123, DRB1*1124, DRB1*1125, DRB1*1126, DRB1*112701, DRB1*112702, DRB1*1128, DRB1*1129, DRB1*1130, DRB1*1131, DRB1*1132, DRB1*1133, DRB1*1134, DRB1*1135, DRB1*1136, DRB1*1137, DRB1*1138, DRB1*1139, DRB1*1140, DRB1*1141, DRB1*1142, DRB1*1143, DRB1*1144, DRB1*1145, DRB1*1146, DRB1*1147, DRB1*1148, DRB1*1149, DRB1*1150, DRB1*1151, DRB1*1152, DRB1*1153, DRB1*1154, DRB1*120101, DRB1*120102, DRB1*120201, DRB1*120202, DRB1*120302, DRB1*1204, DRB1*1205, DRB1*1206, DRB1*1207, DRB1*1208, DRB1*1209, DRB1*1210, DRB1*130101, DRB1*130102, DRB1*130103, DRB1*130201, DRB1*130202, DRB1*130301, DRB1*130302, DRB1*1304, DRB1*1305, DRB1*1306, DRB1*130701, DRB1*130702, DRB1*1308, DRB1*1309, DRB1*1310, DRB1*1311, DRB1*1312, DRB1*1313, DRB1*131401, DRB1*131402, DRB1*1315, DRB1*1316, DRB1*1317, DRB1*1318, DRB1*1319, DRB1*1320, DRB1*1321, DRB1*1322, DRB1*1323, DRB1*1324, DRB1*1325, DRB1*1326, DRB1*1327, DRB1*1328, DRB1*1329, DRB1*1330, DRB1*1331, DRB1*1332, DRB1*1333, DRB1*1334, DRB1*1335, DRB1*1336, DRB1*1337, DRB1*1338, DRB1*1339, DRB1*1340, DRB1*1341, DRB1*1342, DRB1*1343, DRB1*1344, DRB1*1345, DRB1*1346, DRB1*1347, DRB1*1348, DRB1*1349, DRB1*1350, DRB1*1351, DRB1*1352, DRB1*1353, DRB1*1354, DRB1*1355, DRB1*1356, DRB1*1357, DRB1*1358, DRB1*1359, DRB1*1360, DRB1*1361, DRB1*1362, DRB1*1363, DRB1*1364, DRB1*1365, DRB1*140101, DRB1*140102, DRB1*1402, DRB1*140301, DRB1*140302, DRB1*1404, DRB1*140501, DRB1*140502, DRB1*1406, DRB1*140701, DRB1*140702, DRB1*1408, DRB1*1409, DRB1*1410, DRB1*1411, DRB1*1412, DRB1*1413, DRB1*1414, DRB1*1415, DRB1*1416, DRB1*1417, DRB1*1418, DRB1*1419, DRB1*1420, DRB1*1421, DRB1*1422, DRB1*1423, DRB1*1424, DRB1*1425, DRB1*1426, DRB1*1427, DRB1*1428, DRB1*1429, DRB1*1430, DRB1*1431, DRB1*1432, DRB1*1433, DRB1*1434, DRB1*1435, DRB1*1436, DRB1*1437, DRB1*1438, DRB1*1439, DRB1*1440, DRB1*1441, DRB1*1442, DRB1*1443, DRB1*1444, DRB1*1445, DRB1*1446, DRB1*1447, DRB1*1448, DRB1*150101, DRB1*150102, DRB1*150103, DRB1*150104, DRB1*150105, DRB1*150201, DRB1*150202, DRB1*150203, DRB1*1503, DRB1*1504, DRB1*1505, DRB1*1506, DRB1*1507, DRB1*1508, DRB1*1509, DRB1*1510, DRB1*1511, DRB1*1512, DRB1*1513, DRB1*1514, DRB1*1515, DRB1*1516, DRB1*160101, DRB1*160102, DRB1*160201, DRB1*160202, DRB1*1603, DRB1*1604, DRB1*160501, DRB1*160502, DRB1*1607, DRB1*1608.

(49) HLA-DRB2-9

(50) DRB2*0101, DRB3*010101, DRB3*01010201, DRB3*01010202, DRB3*010103, DRB3*010104, DRB3*0102, DRB3*0103, DRB3*0104, DRB3*0105, DRB3*0106, DRB3*0107, DRB3*0108, DRB3*0109, DRB3*0110, DRB3*0111, DRB3*0201, DRB3*020201, DRB3*020202, DRB3*020203, DRB3*020204, DRB3*0203, DRB3*0204, DRB3*0205, DRB3*0206, DRB3*0207, DRB3*0208, DRB3*0209, DRB3*0210, DRB3*0211, DRB3*0212, DRB3*0213, DRB3*0214, DRB3*0215, DRB3*0216, DRB3*0217, DRB3*0218, DRB3*0219, DRB3*030101, DRB3*030102, DRB3*0302, DRB3*0303, DRB4*01010101, DRB4*0102, DRB4*01030101, DRB4*01030102N, DRB4*010302, DRB4*010303, DRB4*010304, DRB4*0104, DRB4*0105, DRB4*0106, DRB4*0107, DRB4*0201N, DRB4*0301N, DRB5*010101, DRB5*010102, DRB5*0102, DRB5*0103, DRB5*0104, DRB5*0105, DRB5*0106, DRB5*0107, DRB5*0108N, DRB5*0109, DRB5*0110N, DRB5*0111, DRB5*0112, DRB5*0113, DRB5*0202, DRB5*0203, DRB5*0204, DRB5*0205, DRB6*0101, DRB6*0201, DRB6*0202, DRB7*010101, DRB7*010102, DRB8*0101, DRB9*0101.

(51) HLA-DQA1

(52) DQA1*010101, DQA1*010102, DQA1*010201, DQA1*010202, DQA1*0103, DQA1*010401, DQA1*010402, DQA1*0105, DQA1*0106, DQA1*0107, DQA1*0201, DQA1*030101, DQA1*0302, DQA1*0303, DQA1*040101, DQA1*040102, DQA1*0402, DQA1*0403N, DQA1*0404, DQA1*050101, DQA1*050102, DQA1*0502, DQA1*0503, DQA1*0504, DQA1*0505, DQA1*060101, DQA1*060102, DQA1*0602.

(53) HLA-DQB1

(54) DQB1*020101, DQB1*020102, DQB1*0202, DQB1*0203, DQB1*030101, DQB1*030102, DQB1*030201, DQB1*030202, DQB1*030302, DQB1*030303, DQB1*0304, DQB1*030501, DQB1*030502, DQB1*030503, DQB1*0306, DQB1*0307, DQB1*0308, DQB1*0309, DQB1*0310, DQB1*0311, DQB1*0312, DQB1*0313, DQB1*0401, DQB1*0402, DQB1*050101, DQB1*050102, DQB1*050201, DQB1*050202, DQB1*050301, DQB1*050302, DQB1*0504, DQB1*060101, DQB1*060102, DQB1*060103, DQB1*0602, DQB1*0603, DQB1*060401, DQB1*060402, DQB1*060501, DQB1*060502, DQB1*0606, DQB1*0607, DQB1*0608, DQB1*0609, DQB1*0610, DQB1*061101, DQB1*061102, DQB1*0612, DQB1*0613, DQB1*0614, DQB1*0615, DQB1*0616, DQB1*0617, DQB1*0618, DQB1*0619, DQB1*0620, DQB1*0621, DQB1*0622, DQB1*0623.

(55) HLA-DPA1

(56) DPA1*010301, DPA1*010302, DPA1*010303, DPA1*0104, DPA1*0105, DPA1*0106, DPA1*0107, DPA1*0108, DPA1*020101, DPA1*020102, DPA1*020103, DPA1*020104, DPA1*020105, DPA1*020106, DPA1*020201, DPA1*020202, DPA1*020203, DPA1*0203, DPA1*0301, DPA1*0302, DPA1*0303, DPA1*0401.

(57) HLA-DPB1

(58) DPB1*010101, DPB1*010102, DPB1*010103, DPB1*0102, DPB1*020102, DPB1*020103, DPB1*020104, DPB1*020105, DPB1*020106, DPB1*0202, DPB1*0203, DPB1*030101, DPB1*030102, DPB1*0302, DPB1*040101, DPB1*040102, DPB1*0402, DPB1*0501, DPB1*0601, DPB1*0801, DPB1*0901, DPB1*1001, DPB1*110101, DPB1*110102, DPB1*1301, DPB1*1401, DPB1*1501, DPB1*1601, DPB1*1701, DPB1*1801, DPB1*1901, DPB1*200101, DPB1*200102, DPB1*2101, DPB1*2201, DPB1*2301, DPB1*2401, DPB1*2501, DPB1*260101, DPB1*260102, DPB1*2701, DPB1*2801, DPB1*2901, DPB1*3001, DPB1*3101, DPB1*3201, DPB1*3301, DPB1*3401, DPB1*3501, DPB1*3601, DPB1*3701, DPB1*3801, DPB1*3901, DPB1*4001, DPB1*4101, DPB1*4401, DPB1*4501, DPB1*4601, DPB1*4701, DPB1*4801, DPB1*4901, DPB1*5001, DPB1*5101, DPB1*5201, DPB1*5301, DPB1*5401, DPB1*5501, DPB1*5601, DPB1*5701, DPB1*5801, DPB1*5901, DPB1*6001, DPB1*6101N, DPB1*6201, DPB1*6301, DPB1*6401N, DPB1*6501, DPB1*6601, DPB1*6701, DPB1*6801, DPB1*6901, DPB1*7001, DPB1*7101, DPB1*7201, DPB1*7301, DPB1*7401, DPB1*7501, DPB1*7601, DPB1*7701, DPB1*7801, DPB1*7901, DPB1*8001, DPB1*8101, DPB1*8201, DPB1*8301, DPB1*8401, DPB1*8501, DPB1*8601, DPB1*8701, DPB1*8801, DPB1*8901, DPB1*9001, DPB1*9101, DPB1*9201, DPB1*9301, DPB1*9401, DPB1*9501, DPB1*9601, DPB1*9701, DPB1*9801, DPB1*9901.

(59) HLA-DMA

(60) DMA*0101, DMA*0102, DMA*0103, DMA*0104.

(61) HLA-DMB

(62) DMB*0101, DMB*0102, DMB*0103, DMB*0104, DMB*0105, DMB*0106.

(63) HLA-DOA

(64) DOA*010101, DOA*01010201, DOA*01010202, DOA*01010203, DOA*010103, DOA*01010401, DOA*01010402, DOA*010105.

(65) HLA-DOB

(66) DOB*01010101, DOB*01010102, DOB*010102, DOB*010201, DOB*010202, DOB*0103, DOB*01040101, DOB*01040102.

(67) MHC Class I

(68) H-2Db, H-2Dd, H-2Dk, H-2Dq, H-2Kb, H-2Kd, H-2Kk, H-2Ld, H-2M3, H-2Ad, H-2Ag7, H-2Ak, H2-Ab, H-2Ed, H-2Ek, H-2Bxk, H-2F, H-2I, H-2P, H-2R, H-2S, H-2Sxd, H-2T4, H-2U.

(69) MHC Class II

(70) I-Ab, I-Ad, I-Ag7, I-Ak, I-Ap, I-Aq, I-Ar, I-As, I-Au, I-Av, I-Ea, I-Eb, I-Ed, I-Ek, I-Es, I-Eu, H-2Q, H-2Qa-2, H-2Qa-2a, Qa-1a, Qa-1b.

(71) The invention is not limited to such MHC and HLA molecules, and can be adapted to newly discovered such molecules, if desired, simply by establishing the reactivity of substances such as peptides with the molecules. This can be readily achieved using known techniques that are standard in the field. Particularly preferred HLA alleles for use with the present invention include the following:

(72) TABLE-US-00011 HLA Class I HLA A HLA B HLA Cw A*6802 B*5801 Cw*1701 A*6801 B*5701 Cw*1601 A*6601 B*5501 Cw*1502 A*3303 B*5201 Cw*1402 A*3301 B*5101 Cw*1203 A*3201 B*5001 Cw*0802 A*310102 B*4901 Cw*0801 A*3002 B*4501 Cw*0704 A*3001 B*4403 Cw*0703 A*2902 B*4402 Cw*0702 A*2608 B*4101 Cw*0701 A*2601 B*4002 Cw*0602 A*2501 B*4001 Cw*0501 A*2402 B*3901 Cw*0401 A*2301 B*3801 Cw*0304 A*1101 B*3701 Cw*0303 A*0302 B*3503 Cw*0202 A*0301 B*3501 Cw*0102 A*0205 B*2705 A*0201 B*1801 A*0101 B*1501 B*1402 B*1401 B*1302 B*0801 B*0705 B*0702

(73) TABLE-US-00012 HLA Class II HLA DPB HLA DQA HLA DQB HLA DRB DPB1*1701 DQA1*0505 DQB1*0604 DRB1*1601 DPB1*1301 DQA1*0501 DQB1*0603 DRB1*1501 DPB1*1001 DQA1*0401 DQB1*0602 DRB1*1401 DPB1*0601 DQA1*0303 DQB1*0503 DRB1*1302 DPB1*0501 DQA1*0302 DQB1*0502 DRB1*1301 DPB1*0402 DQA1*0301 DQB1*0501 DRB1*1201 DPB1*0401 DQA1*0201 DQB1*0402 DRB1*1104 DPB1*0301 DQA1*0104 DQB1*0303 DRB1*1101 DPB1*0201 DQA1*0103 DQB1*0302 DRB1*0801 DPB1*0101 DQA1*0102 DQB1*0301 DRB1*0701 DQA1*0101 DQB1*0202 DRB1*0404 DQB1*0201 DRB1*0401 DRB1*0301 DRB1*0103 DRB1*0102 DRB1*0101

(74) The most preferred alleles according to the invention are the following:

(75) HLA-A*0201, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*2402, HLA-A*3401, HLA-B*0702, HLA-B*0801, HLA-B*1301, HLA-B*27, HLA-B*4002, HLA-B*5101, HLA-Cw*03, HLA-cW*07

(76) HLA-DRB1*0301, HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1501, HLA-DRB1*1104, HLA-DRB1*1101, HLA-DRB4*0101

(77) HLA-DQA1*01, HLA-DQA1*02, HLA-DQA1*05

(78) HLA-DQB1*03, HLA-DQB1*04, HLA-DQB1*05, HLA-DQB1*06

(79) HLA-DPA1*01, HLA-DPA1*02

(80) HLA-DPB1*02, HLA-DPB1*04

(81) The invention will now be described by way of example only, with reference to the following specific embodiments.

EXAMPLES

Preparation of Arthropod Saliva Protein Fractions

(82) In order to determine the effect of specific sequences of the invention, their immunogenicity may be tested against various arthropod saliva protein fractions. Those sequences that cause a vertebrate to produce immune system cells that recognise at least one epitope in specific saliva protein fractions are useful in the vaccines of the present invention.

(83) The saliva protein fractions can easily be isolated using standard laboratory techniques, which are well known to the skilled person. Any arthropod saliva protein fractions may be used, since the inventors have determined that it is the mass of the protein fraction that is important. The fractions of mass 40 kDa or less, 30 kDa or less, preferably from 20-40 kDa, and more preferably 20 kDa or less, are particularly useful.

(84) The following protocol is provided to exemplify the protein fraction against which candidate sequences may be tested. It utilises saliva from anopheles gambiae mosquitoes, although any arthropod saliva may be employed.

(85) Anopheles gambiae salivary gland pairs (SGP) were dissected from female mosquitoes colonies. Fifteen SGPs were collected in 20 l of PBS and lysed by adding 5 l of 5SDS-PAGE sample buffer containing 0.25% 2--ME. After vortexing and boiling for 5 min, the protein mixture was loaded on a Novex 4-20% gradient Tris-Glycine gel (INVITROGEN). The gel was then silver stained and photographed (see FIG. 9).

(86) The dashed arrows indicate the position of the cut off points for selection of SGP fractions that may be used for immunisation and study (i.e. <20 kDa, 20-40 kDa, 40-80 kDa and >80 kDa). The solid arrow indicates the position of the cut off point for selection of SGP fractions of <30 kDa and >30 kDa.

(87) Identifying Candidate Protein Samples from Mosquito Saliva

(88) Taking a similar approach to the exemplary protocol outlined above, several saliva protein samples were prepared for study.

(89) Salivary glands (SG) were dissected from female Anopheles gambiae mosquitoes and stored in PBS at 70 C. until use.

(90) For gel analysis, SGs were lysed by freeze-thawing, Novex IEF Sample Buffer pH 3-10 (Invitrogen) added and the resulting material analysed in a Novex IEF Gel (Invitrogen). The gel was then fixed with 12% TCA, washed three times in water and stained with Comassie Blue. The resulting gel is shown in FIG. 1.

(91) After staining and destaining, the IEF gel was incubated in 20% ethanol for 10 minutes and the gel strip containing the lane with the SG proteins cut out. This gel strip was equilibrated for 5 min in 2SDS-PAGE sample buffer containing 20% ethanol, rinsed twice in SDS-PAGE sample buffer and loaded in the single well of a Novex 4-20% Tris-Glycine Gel (Invitrogen). The resulting gel is shown in FIG. 2.

(92) The gel containing the separated SG proteins was finally stained using the Proteosilver stain kit (Sigma) according to the manufacturer's instructions.

(93) From the gel, four groups of proteins (designated compounds 1, 2, 3 and 4) were identified for analysis:

(94) Compound 1 Salivary Gland Protein Fraction <20 kDa

(95) Compound 2 Salivary Gland Protein Fraction 20 kDa<X<40 kDa

(96) Compound 3 Salivary Gland Protein Fraction 40 kDa<X<80 kDa

(97) Compound 4 Salivary Gland Protein Fraction >80 kDa

Experiment 1

Aims

(98) (A) Establish efficacy of candidates in protecting animals against challenge by bite of infected mosquitoes (i.e. Plasmodium yoelii nigeriensis infected Anopheles gambiae)

(99) (B) Establish cross-reactivity level of the anti-mosquito response induced by candidates across different species of mosquitoes (e.g. Anopheles gambiae and Anopheles stephensi).

(100) (C) Establish efficacy of candidates in preventing infection of mosquitoes (Anopheles gambiae and Anopheles stephensi) by Plasmodium yoelii nigeriensis after biting immunised and infected mice.

(101) Candidate Compounds

(102) The compounds selected were those identified above:

(103) Compound 1 Salivary Gland Protein Fraction <20 kDa

(104) Compound 2 Salivary Gland Protein Fraction 20 kDa<X<40 kDa

(105) Compound 3 Salivary Gland Protein Fraction 40 kDa<X<80 kDa

(106) Compound 4 Salivary Gland Protein Fraction >80 kDa

(107) Strain and Number of Animals Involved:

(108) CD1 mice are used. There were five experimental groups (1, 2, 3, 4 and 5) with group 1 being the negative control group and groups 2-5 the test groups, each group having nine (9) animals.

Experimental Protocol

(109) Day 1: 4 groups were immunised (groups 2, 3, 4 and 5) of 9 CD1 mice each (N=49=36) with subcutaneous doses of candidate vaccine compounds (Group 2 with compound 1, Group 3 with compound 2, and so on).

(110) Day 14: All animals were boosted with the same doses of candidate vaccine candidate vaccine compounds (Group 2 with compound 1, Group 3 with compound 2, and so on).

(111) Day 21: All animals were test bled. Samples were stored frozen (20 C.) until collection. Each group was split into further subgroups: A5 animals, B4 animals.

(112) TABLE-US-00013 Group 1A - 5 animals Group 1B -4 animals Group 2A - 5 animals Group 2B - 4 animals Group 3A - 5 animals Group 3B - 4 animals Group 4A - 5 animals Group 4B - 4 animals Group 5A - 5 animals Group 5B - 4 animals

(113) Subgroups A

(114) Day 28: All animals in subgroups A were challenged via bite of 5-9 infected mosquitoes (i.e. Plasmodium yoelii nigeriensis infected Anopheles gambiae) in the belly area. All animals were maintained until parasitemia is first established or for a maximum of 6 weeks after challenge with infected mosquitoes. All animals were killed by exsanguination and the sera samples were stored frozen (20 C.) until collection.

(115) Subgroups B

(116) Day 28: All animals in subgroups B were used to feed (in the belly area) the following number of fresh (uninfected) mosquitoes:

(117) 5-10 Anopheles gambiae AND 5-10 Anopheles stephensi

(118) All these mosquitoes were tested for:

(119) 1. Survival over an 8 day period.

(120) 2. Number of eggs laid.

(121) 3. Number of eggs produced.

(122) 4. Number of adults (F1) produced from the eggs laid.

(123) Day 32: All animals in subgroups B were infected with Plasmodium yoelii nigeriensis by direct IV inoculation of parasites.

(124) Day 32: Once active malaria infection had been identified in all (or at least 75%) animals in Group B, all infected animals were used to feed (in the belly area) large numbers (>10 per mice) of both fresh Anopheles gambiae AND fresh Anopheles stephensi.

(125) All these mosquitoes were tested for: 1. Survival over an 8 day period. 2. Number of mosquitoes with malarial parasites in their salivary glands amongst those who survived the required incubation period (17 days). 3. Number of eggs laid. 4. Number of eggs produced. 5. Number of adults (F1) produced from the eggs laid.

(126) After mosquito feeding, all animals were killed by exsanguination and the sera samples were stored frozen (20 C.) until collection.

(127) Any volume of the experimental compound remaining at the end of the study was stored frozen (20 C.) until collection.

(128) Results

(129) Graphical representations of the results of the experiments are shown in FIGS. 3A-3I and 4. The first set of Figures (FIG. 3x) show data on the effect of the vaccine on the fecundity of mosquitoes, for each of the Groups 1-5, as follows:

(130) TABLE-US-00014 3A: Percent that fed 3B: Mean no eggs produced 3C: Mean no eggs laid 3D: Percent hatch rate 3E: Mean no larvae 3F: Mean no pupae 3G: Percent pupation 3H: Percent emergence 3I: Mean no adults

(131) The numerical data underpinning each of these graphical representations A-I are set down respectively (column A for FIG. 3A, and so on) in Table 2 below:

(132) TABLE-US-00015 TABLE 2 Data for FIGS. 3A-3I Mean Std. Error A B C D E Group 1 82.7 4 29.6 5 37.5 4.6 20.1 3.9 54.6 4.7 Group 2 66.7 6.5 17.1 3.8 12 3 9.7 2.4 51.9 7.4 Group 3 81.2 3.4 19.1 4.6 16 3.7 12.9 4 44.3 8.7 Group 4 73.7 4.9 24.1 3.9 26.4 3.9 15 2.8 56.6 6.6 Group 5 75.7 4.5 30.1 4.1 32.4 5 16.9 2.5 55 4.6 Mean Std. Error F G H I Group 1 15.6 3 82.9 3 14 2.8 92.4 2.6 Group 2 7.8 1.8 86.1 5.7 6.9 1.8 80 7.8 Group 3 12.6 3.7 77.7 7.3 12.5 3.6 92 1.8 Group 4 12.3 2.1 88.5 3.3 10.8 1.9 88.8 2.4 Group 5 14.4 2.2 88.4 2.7 12.1 1.8 84.8 3.7

(133) The data set out in Tables 3A-3I below show the p-values obtained using a Mann-Whitney non-parametric statistical analysis of the above data.

(134) TABLE-US-00016 TABLE 3A P values for percent that fed Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.10504 Group 3 0.41727 0.14813 Group 4 0.14813 0.26543 0.10504 Group 5 0.23235 0.23235 0.26543 0.5

(135) TABLE-US-00017 TABLE 3B P values for mean no eggs produced Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 4.6E05 Group 3 0.00042 0.1612 Group 4 0.04038 0.0032 0.01602 Group 5 0.17836 0.0018 0.00551 0.22172

(136) TABLE-US-00018 TABLE 3C P values for mean no eggs laid Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.09451 Group 3 0.07626 0.49308 Group 4 0.27329 0.09416 0.10891 Group 5 0.30616 0.01171 0.00879 0.15145

(137) TABLE-US-00019 TABLE 3D P values for percent hatch rate Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.46767 Group 3 0.19155 0.29445 Group 4 0.36699 0.33803 0.14632 Group 5 0.40058 0.47907 0.15041 0.40187

(138) TABLE-US-00020 TABLE 3E P values for mean no larvae Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.04897 Group 3 0.07371 0.45038 Group 4 0.29801 0.06197 0.11695 Group 5 0.4499 0.0164 0.06524 0.23691

(139) TABLE-US-00021 TABLE 3F P values for mean no pupae Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.06224 Group 3 0.28662 0.22088 Group 4 0.44057 0.04081 0.35186 Group 5 0.40546 0.01118 0.17373 0.25066

(140) TABLE-US-00022 TABLE 3G P values for percent pupation Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.11277 Group 3 0.3664 0.13511 Group 4 0.11848 0.50798 0.07764 Group 5 0.10627 0.41971 0.08354 0.37388

(141) TABLE-US-00023 TABLE 3H P values for percent emergence Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.06876 Group 3 0.12815 0.25697 Group 4 0.059 0.33712 0.2767 Group 5 0.02006 0.4853 0.18532 0.2943

(142) TABLE-US-00024 TABLE 3I P values for mean no adults Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 0.04391 Group 3 0.47127 0.06735 Group 4 0.3981 0.03908 0.47468 Group 5 0.46488 0.0123 0.31328 0.24603

(143) FIG. 4 shows the percent survival of the mosquitoes in each of the five groups, over a period of 8 days. The number of mosquitoes in each group at day zero was as follows:

(144) TABLE-US-00025 Group 1 (control): 100 Group 2: 89 Group 3: 100 Group 4: 99 Group 5: 98

(145) The data relevant to the graphical representation of FIG. 4 is set out in Table 4 below:

(146) TABLE-US-00026 TABLE 4 Data for FIG. 4 Mean Std. Error Day 0 Day 1 Day 2 Day 3 Day 4 Group 1 100 0 86 4.2 77 5 73 5.2 61 4.6 Group 2 100 0 53.4 5.5 40.4 5.3 36.4 4.9 28 5.2 Group 3 100 0 67 4.5 51 4.7 44 4.9 36 4.4 Group 4 100 0 78 5.1 62 5.7 60 5.6 51 4.9 Group 5 100 0 73 5.6 53.4 6.7 48.4 6.3 41.4 5.6 Mean Std. Error Day 5 Day 6 Day 7 Day 8 Group 1 58 4.8 56 4.4 53 4.5 53 4.5 Group 2 23.7 5 21.4 4.8 20 4.8 20 4.8 Group 3 30 4.1 28 3.7 28 3.7 28 3.7 Group 4 46 5 46 5 43 5.2 43 5.2 Group 5 36.7 5 33.7 5.3 29.4 5 29.4 5

(147) The data set out in Table 5(i)-5(viii) below show the p-values obtained using a Mann-Whitney non-parametric statistical analysis of the above data.

(148) TABLE-US-00027 TABLE 5(i) P values for day 1 Day 1 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 2.6E05 Group 3 0.00217 0.0326 Group 4 0.13228 0.0007 0.0387 Group 5 0.0526 0.0071 0.1588 0.2738

(149) TABLE-US-00028 TABLE 5(ii) P values for day 2 Day 2 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 1.1E05 Group 3 0.0003 0.0676 Group 4 0.0274 0.00453 0.06759 Group 5 0.0049 0.07833 0.44598 0.166

(150) TABLE-US-00029 TABLE 5(iii) P values for day 3 Day 3 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 1.1E05 Group 3 0.0002 0.20756 Group 4 0.04955 0.00253 0.0213 Group 5 0.00311 0.07692 0.26734 0.10538

(151) TABLE-US-00030 TABLE 5(iv) P values for day 4 Day 4 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 3.8E05 Group 3 0.00032 0.12615 Group 4 0.09515 0.00175 0.01525 Group 5 0.0065 0.04759 0.24854 0.09848

(152) TABLE-US-00031 TABLE 5(v) P values for day 5 Day 5 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 2.6E05 Group 3 6.9E05 0.14515 Group 4 0.07976 0.00198 0.00944 Group 5 0.00393 0.03955 0.14962 0.09848

(153) TABLE-US-00032 TABLE 5(vi) P values for day 6 Day 6 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 1E05 Group 3 2.3E05 0.12817 Group 4 0.12022 0.00081 0.00393 Group 5 0.0034 0.05802 0.22172 0.0558

(154) TABLE-US-00033 TABLE 5(vii) P values for day 7 Day 7 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 2.3E05 Group 3 8.4E05 0.07147 Group 4 0.13648 0.00175 0.01315 Group 5 0.00132 0.09032 0.40795 0.03713

(155) TABLE-US-00034 TABLE 5(viii) P values for day 8 Day 8 Compound 1 Compound 2 Compound 3 Compound 4 Group 1 Group 2 2.3E05 Group 3 8.4E05 0.07147 Group 4 0.13648 0.00175 0.01315 Group 5 0.00132 0.09032 0.40795 0.03713

Experiment 2

Induction of Cytokine Production by Polypeptide Antigens immunisation

(156) Peptides and Recombinant Proteins

(157) From experiment 1, the most effective fraction was studied further, to identify polypeptides for that may be employed in vaccines. The utility of the peptides of the present invention was determined using the following protocol.

(158) Immunisations

(159) All the polypeptides under study (antigen preparations) are synthesised by Fmoc chemistry.

(160) Six to ten week old C57BL/6 mice are immunised subcutaneously with a 200 l dose of the antigen preparation per mouse. In the test group, each dose of the antigen preparation contains an equimolar mixture of the peptides (10 nmol each) prepared in adjuvant (Sigma) according to the manufacturer's instructions. In the control group, each dose of the antigen preparation contains an equivalent dose of a non-relevant polypeptide prepared in IFA (Sigma) according to the manufacturer's instructions (NRP preparation).

(161) On day 15 post-immunisation, all animals receive a booster immunisation using the same doses and route of delivery as originally.

(162) Finally, on day 20 all animals are culled and their spleens and sera are collected.

(163) Cytokine ELISA

(164) Mouse spleens belonging to the same experimental group are pooled, gently pressed through cell strainers and red blood cells removed by treatment with red cell lysis buffer (nine parts 0.16 M NH.sub.4Cl and one part of 0.17 M Tris, pH 7.2). Splenocyte suspensions from each experimental group are plated in 96-well plates in quadruplicate at a density of 410.sup.6 cells/well in IMDM medium (Invitrogen) supplemented with 0.02 mM -mercaptoethanol (Sigma), 50 IU/50 mg/ml of penicillin/streptomycin (Sigma) and 10% FCS (Sigma) and containing each of the polypeptide antigens under study (2 M). After 3 days incubation at 37 C., the supernatant is collected and analysed for IFN- and IL-4 by a sandwich cytokine ELISA according to the manufacturer's protocol (Pharmingen). The lower detection limits for the assay are 9.77 pg/ml for IL-4 and 39.06 g/ml for IFN-.

(165) IgG2a Specific ELISA

(166) Microtiter ELISA 96-well plates (Becton-Dickinson) are coated with 2 M of each experimental polypeptide in PBS. After overnight at incubation at 4 C., plates are washed twice in PBST (PBS containing 0.05% of Tween 20) and wells blocked with 1% BSA Fraction V in PBST. After 1 h incubation, plates are washed thrice in PBST and a range of dilutions of test and control sera in PBST added to the wells. After 2 h incubation, plates are washed six times in PBST, and primary anti-mouse-Ig2a sera are added to all wells. After 1 h incubation, plates are washed six times in PBST, and anti-primary anti-mouse-Ig2a sera added to all wells. After 1 h incubation, plates are washed seven times with in PBST and TMB substrate to all wells. After 20-30 minutes incubation, the reaction is stopped with HCl and the absorbance at 450 nm is read.

(167) Statistical Analysis

(168) Statistically significant differences in the IFN- response to different antigens between the test and control groups are established through non-parametric Mann-Whitney analysis of the samples. Differences are considered statistically significant if the p value is below 0.05.

Experiment 3

Assessment of the Immune Response to Various Polypeptides

(169) The following polypeptides were investigated: SEQ ID 20, SEQ ID 28, SEQ ID 30, SEQ ID 31, SEQ ID 32 and SEQ ID 35. These peptides were mixed together to form a candidate vaccine for testing (called the AGS peptide mix).

(170) The type and level of the immune response induced by vaccination with these peptides was assessed according to the protocol shown below: Day 1: Immunise 2 groups of 4 CD1 mice each with the following subcutaneous doses of candidate vaccine products: Non-relevant-peptide (NRP) mix (10 nmol each)+ISA-51 AGS peptide mix (10 nmol each)+ISA-51 Day 15: Boost all animals with the same doses of candidate vaccine products. Day 21: Terminally bleed all animals. Harvest spleens individually and test for IFN-gamma reactivity to: Individual AGS peptides (2 M each) AGS mix (0.5 M and 2 M each) Con A (7.5 g/ml) Blank Post-Day 21: Test all sera for reactivity against AGS peptides.

(171) Results

(172) IFN-gamma production following 96 hour stimulation in vitro with the antigens is indicated in the graph in FIG. 5.

(173) IFN-gamma responses to SEQ ID 28, SEQ ID 30 and SEQ ID 35 as well as to the AGS-mix preparation are statistically significant (p<0.05).

(174) SEQ ID 20, SEQ ID 31 and SEQ ID 32 induce a higher response in the AGS-mix immunised animals, but they also appear to be stimulatory, in a non specific fashion, to the splenocytes of NRP-mix immunised mice.

(175) The total Ig response in sera to the antigens indicated in the graph in FIG. 6. Total Ig responses to SEQ ID 20 and SEQ ID 30 are statistically significant (p<0.05).

Experiment 4

Challenge Study Following AGS Peptide Mix Immunisation

(176) To test the capacity of the AGS-mix preparation to confer protection against natural malaria infection, CD1 mice were immunised and challenged according to the protocol below: Day 0: Test bleed all animals. Retain samples for further analysis. Day 1: Immunise 2 (two) groups of 8 CD1 mice each with the following subcutaneous doses of candidate vaccine products: Non-relevant-peptide (NRP) mix (10 nmol each)+ISA-51 AGS peptide mix (10 nmol each)+ISA-51 Day 7: Test bleed all animals. Retain samples for further analysis. Day 14: Boost animals with the same doses of candidate vaccine products. Day 21: Test bleed all animals. Retain samples for further analysis. Day 28: All animals are challenged via bite of 8 infected mosquitoes (i.e. Plasmodium yoelii nigeriensis infected Anopheles gambiae) in the belly area. All animals are maintained until parasitemia is first established or for a maximum of 6 weeks after challenge with infected mosquitoes. Day 70 (max): Terminally bleed all animals.

(177) Results

(178) Total Ig response in sera to the AGS-mix at day 21 is indicated in the graph in FIG. 7.

(179) One animal in the AGS-mix immunised group showed a significantly lower Total Ig response than the remaining animals in the group (<50% average total Ig response in the AGS-mix group).

(180) On the day of challenge, one animal in the NRP-mix immunised group and two animals in the AGS-mix immunised group could not be challenged due to a shortage of infectious mosquitoes.

(181) Of the animals that were challenged, those in the AGS-mix immunised group showed an increased survival rate than those in the control NRP-mix immunised group (see FIG. 8). The one animal that died in the AGS-mix immunised group was the same one that had failed to develop a strong antibody response to the AGS preparation.