TOXOPLASMA GONDII VACCINES AND THEIR USE
Disclosed herein are polynucleotides encoding multi-epitope polypeptides and assemblies thereof, and their use for treating or limiting Toxoplasma gondii infection.
1 . An isolated polynucleotide encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises
(a) a plurality of first peptide domains wherein each first peptide domain is a CD8 + T cell eliciting epitope, including but not limited to FLSLSLLVI (SEQ ID NO: 1), FMIAFISCFA (SEQ ID NO: 2), FVIFACNFV (SEQ ID NO: 3), FMIVSISLV (SEQ ID NO: 4), FLLGLLVHV (SEQ ID NO: 5), FLTDYIPGA (SEQ ID NO: 6), ITMGSLFFV (SEQ ID NO: 7), GLAAAVVAV (SEQ ID NO: 8), VLLPVLFGV (SEQ ID NO: 9), FAAAFFPAV (SEQ ID NO: 10), VVFVVFMGV (SEQ ID NO: 11), FMGVLVNSL (SEQ ID NO: 12), FLVPFVVFL (SEQ ID NO: 13), STFWPCLLR (SEQ ID NO: 14), SSAYVFSVK (SEQ ID NO: 15), KSFKDILPK (SEQ ID NO: 16), AVVSLLRLLK (SEQ ID NO: 17), and AMLTAFFLR (SEQ ID NO: 18)); and
(b) one or more second peptide domains, wherein each second peptide domain is a CD4+ epitope, including but not limited to AKFVAAWTLKAAA (SEQ ID NO: 19), AVEIHRPVPGTAPPS (SEQ ID NO: 20), IRLLASLHH (SEQ ID NO: 39), LIRLLASLH (SEQ ID NO: 40), LTLQLIRLL (SEQ ID NO: 41), VIEEFNRI (SEQ ID NO: 42), LQLIRLLAS (SEQ ID NO: 43), IDVVIEELF (SEQ ID NO: 44),or a derivative thereof.
2 . The isolated polynucleotide of claim 1 , wherein (i) the plurality of first peptide domains comprises at least 5 peptide domains, wherein the at least 5 peptide domains include KSFKDILPK (SEQ ID NO: 16), STFWPCLLR (SEQ ID NO: 14), AVVSLLRLLK (SEQ ID NO: 17), SSAYVFSVK (SEQ ID NO: 15), AMLTAFFLR (SEQ ID NO: 18); and/or (ii) the one or more second peptide domains include AKFVAAWTLKAAA (SEQ ID NO: 19) or a derivative thereof.
3 . (canceled)
4 . The isolated polynucleotide of claim 1 , wherein some or all of the plurality of first peptide domains are separated from the other first peptide domains peptides by a spacer of 1 or more amino acid residues and/or by the one or more second peptide domains.
5 . The isolated polynucleotide of claim 4 , wherein the spacer is selected from the group consisting of N, K, NA 1-3 (SEQ ID NO: 21), KA 1-3 (SEQ ID NO: 22), and GPGPG (SEQ ID NO: 23).
6 . The isolated polynucleotide of claim 1 , wherein the chimeric polypeptide comprises the amino acid sequence
(SEQ ID NO: 24)
AVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAA
KFVAAWTLKAAAKSTFWPCLLR
7 . The isolated polynucleotide of claim 1 , wherein the chimeric polypeptide further comprises a third peptide domain comprising a peptide capable of promoting self-assembly/multimerization (2, 3, 4, 5, 6, or more assembled copies) of the polypeptide.
8 . (canceled)
9 . The isolated polynucleotide of claim 7 , wherein the third peptide domain comprises the sequence selected from the group consisting of:
(SEQ ID NO: 25)
WEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWM;
(SEQ ID NO: 26)
RLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEE
L;
or both.
10 . The isolated polynucleotide of claim 7 , wherein the chimeric polypeptide comprises the amino acid sequence SEQ ID NOS:27-28. A11 (residues in parentheses are optional)
(SEQ ID NO: 27)
((MGDKHHHHHHHHHH))KDGSDKGSWEEWNARWDEWENDWNDWREDWQA
WRDDWARWRATWMGGRLLSRLERLERRNEELRRLLQLIRHENRMVLQFVR
ALSMQNAELERRLEELARGMAQVINTNSLSLLTQNNLNRSQSALGTAIER
LSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGA
LNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQ
FNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNVQQEYES
DDAVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKA
AAKFVAAWTLKAAAKSTFWPCLLRDSDSDTENPLQRIDAALAQVDALRSD
LGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQA
GTSVLAQANQVPQNVLSLLR
Tox-all (residues in parentheses are optional)
(SEQ ID NO: 28)
(MGDDHHHHHHHHHH)WFMGVLVNSLQDITMGSLFFVQDFMIVSISLVQD
GLAAAVVAVQDLPQFATAATRDSPASGRYIQQMLDQRCQEIAAELCQSGL
RKMCVPSSRIVARNAVGITHQNTLQWRCFDTASLLESNQENNGVNCVDDC
GHTIPCPGGVHRQNSNHATRHEILSKLVEEGVQRFCSPYQASANKYCNDK
FPGTIARRSKGFGNNVEVAWRCYEKASLLYSVYAECASNCGTTWYCPGGR
RGTSTELDKRHYTEEEGIRQAIGSVDSPCSEVEVCLPKDENPPLCLDESG
QISRGSWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWMGGRLLSR
LERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEELARGM
AQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIA
NRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSAN
STNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGAN
DGETIDIDLRQINSQTLGLDQLNVQQEYESDDAVVSLLRLLKNAMLTAFF
LRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWP
CLLRDSDSDTENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVN
NLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR
11 . The isolated polynucleotide of claim 1 , wherein the chimeric polypeptide further comprises a secretory signal, including but not limited to the murine Igκ-chain signal sequence.
12 . The isolated polynucleotide of claim 11 , wherein the secretory signal is present at the N-terminus of the chimeric polypeptide.
13 . A recombinant expression vector, comprising the isolated polynucleotide of claim 1 operatively linked to a control sequence.
14 . (canceled)
15 . The recombinant expression vector of claim 13 , wherein the vector comprises a gene encoding a self-amplifying RNA operatively linked to the polynucleotide.
16 . The recombinant expression vector of claim 15 , wherein the self-amplifying RNA comprises the nucleic acid sequence of SEQ ID NO:29
(SEQ ID NO: 29)
GAAUUUGCUGCGACGAUGGGUAUGCAAGUCCAGAUCCAGAGCCUGUUCCU
GCUGCUGCUGUGGGUGCCGGGUUCACGCGGUAUGGCGGUGGUUAGCCUGC
UGCGUCUGCUGAAAAACGCCAUGCUGACCGCAUUUUUCCUGCGCAAUGCG
GCCGCAAAGAGUUUCAAGGAUAUCCUGCCGAAAAAGGCUGCGGCCAGCUC
UGCGUAUGUCUUUUCCGUGAAAAAGGCAGCUGCGAAAUUCGUUGCUGCCU
GGACCCUGAAAGCUGCCGCUAAAUCGACGUUCUGGCCGUGUCUGCUGCG
U.
17 . The recombinant expression vector of claim 15 , wherein the self-amplifying RNA is encoded by the nucleic acid sequence of SEQ ID NO:30.
(SEQ ID NO: 30)
CATGGGTATGCAGGTCCAGATTCAGTCACTCTTTCTCCTCCTCCTCTGGG
TCCCCGGTAGCCGGGGTATGGCCGTGGTCAGCCTGCTCAGGCTGCTCAAG
AACGCCATGCTGACCGCTTTCTTTCTCAGAAATGCCGCTGCAAAGTCTTT
CAAAGACATCCTGCCCAAGAAAGCCGCTGCAAGCTCCGCCTACGTGTTCA
GTGTCAAGAAAGCCGCTGCAAAATTTGTGGCC
18 . A chimeric polypeptide comprising the chimeric polypeptide encoded by the polynucleotide of claim 1 .
19 . (canceled)
20 . A chimeric RNA, comprising the RNA expression product of the polynucleotide claim 1 .
21 . (canceled)
22 . A pharmaceutical composition, comprising:
(a) the chimeric polynucleotide of claim 1 ; and
(b) a pharmaceutically acceptable carrier.
23 . The pharmaceutical composition of claim 22 , wherein composition comprises an adjuvant, such as a TLR4 ligand.
24 .- 26 . (canceled)
27 . The pharmaceutical composition of claim 22 , wherein the composition comprises a vaccine, such as a vaccine formulated for injection or electroporation.
28 . A method for treating or limiting Toxoplasma gondii infection, comprising administering to a subject at risk of Toxoplasma gondii infection an amount effective to treat or limit the infection of the chimeric polynucleotide of claim 1 .
29 .- 31 . (canceled)