Toxoplasma gondii vaccines and their use

Abstract

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)