Modified human rotaviruses and uses therefor

Abstract

The present disclosure relates generally to the field of viral vaccines. Particularly, the present disclosure provides a modified Vero-adapted human rotavirus strain and a culturing method to produce high titer virus, a rotavirus vaccine, vaccination protocols and diagnostics and prognostic assays.

1 . A method for culturing a strain of rotavirus (RV), said method comprising subjecting an aliquot or inoculum of RV to activation by a trypsinization step comprising administering trypsin at greater than 3 times the standard concentration of trypsin used for activation of RV for infection into cells of 5-15 μg/ml and culturing the activated RV in Vero cells or a Vero cell-derived cell line for a time and under conditions sufficient for the RV to undergo a genetic modification resulting in an amino acid change in one or more of VP1, VP3, VP4, NSP4 and/or NSP5/6 and/or nucleotide change in one or more of VP1, VP3, VP4, NSP2, NSP4 and/or NSP5/6. 
     
     
         2 . The method of  claim 1  wherein the RV strain is human RV3. 
     
     
         3 . The method of  claim 1  comprising the additional step of harvesting the RV. 
     
     
         4 . The method of  claim 1  wherein the trypsin is porcine, ruminant animal or recombinant trypsin. 
     
     
         5 . The method of  claim 4  wherein the ruminant animal trypsin is bovine, ovine, caprine or  camelus  trypsin. 
     
     
         6 . The method of  claim 1  wherein the amino acid or nucleotide change is compared to MA104-adapted RV. 
     
     
         7 . The method of  claim 1  wherein the amino acid or nucleotide change is in a codon in NSP4 selected from 53, 76, 85, 162 and 165, a codon in NSP5/6 selected from 46 and 112, a codon in VP1 selected from 886, a codon in VP3 selected from 643 and 785, and a codon in VP4 selected from 43, 374, 385, 388, 442 and 756 and/or a nucleotide in NSP2 selected from 433, a nucleotide in NSP4 selected from 197, 267, 294, 525 and 534, a nucleotide in NSP5/6 selected from 158, 165 and 355, a nucleotide in VP1 selected from 2692, a nucleotide in VP3 selected from 343, 1366, 1744, 1976 and 2402 and/or a nucleotide in VP4 selected from 136, 369, 1130, 1162, 1171, 1334 and 2276. 
     
     
         8 . The method of  claim 1  wherein the amino acid or nucleotide change is selected from the list provided in Table 2 or Table 5. 
     
     
         9 . A method for generating an attenuated RV3 in Vero cells, said method comprising subjecting an aliquot or inoculum of RV3 to activation by a trypsinization step comprising administering trypsin at greater than 3 times the standard concentration of trypsin used for activation of RV3 for infection into cells of 5-15 μg/ml and culturing the activated RV3 and culturing the activated RV3 with Vero cells or a Vero cell-derived cell line for a time and under conditions sufficient for a genetic modification to occur selected from the list provided in Table 2 or Table 5 compared to MA104-adapted RV3. 
     
     
         10 . The method of  claim 9  wherein the trypsin is porcine, ruminant animal or recombinant trypsin. 
     
     
         11 . The method of  claim 10  wherein the ruminant animal trypsin is bovine, ovine, caprine or  camelus  trypsin. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . A method for vaccinating a human subject against RV infection, said method comprising administering to said subject an effective amount of the strain of rotavirus of  claim 9  for a time and under conditions sufficient to generate an immune response against RV infection, which immune response is preferably a protective immune response. 
     
     
         16 . A method for treating a human subject with an RV infection or at risk of developing an RV infection or re-infection said method comprising administering to said subject an effective amount of the strain of rotavirus of  claim 9  for a time and under conditions sufficient to generate an immune response against RV infection. 
     
     
         17 . An isolated antibody to the attenuated RV3 in Vero cells of  claim 13 . 
     
     
         18 . A method for detecting or monitoring the attenuated RV3 in Vero cells of  claim 13  said method comprising screening for a genetic modification resulting in an amino acid change in one or more of VP1, VP3, VP4, NSP4 and/or NSP5/6 and/or nucleotide change in one or more of VP1, VP3, VP4, NSP2, NSP4 and/or NSP5/6. 
     
     
         19 . The strain of rotavirus (RV) produced by the method of  claim 1 . 
     
     
         20 . The attenuated RV3 in Vero cells produced by the method of  claim 10 .