Rational design of rotavirus NSP1 and NSP4 to improve the safety profile of rotavirus vaccine candidates

Abstract

Rotaviruses cause acute gastroenteritis and are the leading cause of diarrhoea-related hospitalisations and deaths worldwide in children under the age of 5. Currently available live-attenuated rotavirus vaccines have significantly alleviated the burden of disease. However, these vaccines are less effective in Sub-Saharan Africa and South-East Asia countries, and there is a need for a better vaccine in some regions. As novel live-attenuated vaccine candidates are being developed in our research group, we must ensure that the vaccines are safe. The non-structural proteins NSP4 and NSP1 contribute to viral pathogenesis. NSP4 is an enterotoxin that increases intracellular calcium levels leading to diarrhoea, while NSP1 inhibits the innate immune response. Understanding how to attenuate these proteins without affecting their other functions in the replication cycle could be the key to increasing the safety profile of novel vaccine candidates. The plasmid-only based reverse genetics system has made it possible to study rotavirus proteins and the impact of altering specific domains. This study's focus was to rationally design NSP1 and NSP4 to rescue rotaviruses that cannot inhibit the nuclear accumulation of phosphorylated STAT1 and STAT2 or increase intracellular calcium levels. Controls are needed to test the vaccine candidates developed in our research group to ensure that they are safe. The attenuated and virulent NSP4 of the porcine OSU rotavirus can be used as such controls. The proteins were previously expressed using the baculovirus expression system, and it was hypothesised that codon optimising the ORFs would increase protein yield. Two pFastBac-HTA donor plasmids containing the Sf9 insect cell codon optimised OSU-a NSP4 and OSU-v NSP4 ORF were designed and used in the Bac-to-Bac baculovirus expression system to generate recombinant baculoviruses expressing His-tagged OSU-a NSP4 and OSU-v NSP4. Although there were signs of CPE in cells infected with the recombinant baculoviruses, no expression of His-tagged OSU-a NSP4 and OSU-v NSP4 could be observed using SDS-PAGE or immuno-fluorescence monolayer assay. Studies of the amino acid sequences of OSU-a NSP4 and OSU-v NSP4 showed that there were six amino acid differences between the proteins. Three of the six alterations in OSU-a NSP4 clustered in the signal-transducing domain. Altering these amino acids in OSU-v NSP4 attenuated its ability to increase intracellular calcium and cause diarrhoea. The plasmid-only based reverse genetics system was used to investigate if OSU-a NSP4 could be incorporated into SA11-L2 and if altering aa 135-141 of SA11-L2 to those of OSU-a NSP4 would attenuate the virus. Rotaviruses with OSU-a NSP4 and SA11-L2 NSP4 with aa 135-141 of OSU-a NSP4 were successfully rescued. Replication kinetics showed that the presence of OSU-a NSP4 or alterations in SA11-L2 NSP4 did not hinder viral replication, and both rSA11_OSU-a_NSP4 and rSA11_OSU-a_135-141 grew at a rate similar to rSA11-L2. The calcium flux assay iii showed that rSA11_OSU-a_NSP4 could increase intracellular calcium in infected cells, while rSA11_OSU-a_135-141 was partially attenuated. The next step will be to investigate which alternations can be incorporated into the enterotoxin and viroporin domain of GS10(NSP4) of rSA11_OSU-a_135-141 to attenuate the virus completely. NSP1 anchors to the cytoskeleton using its cytoskeleton domain (aa 84-176) and then inhibits the production of interferons and nuclear accumulation of phosphorylated STAT1 and STAT2. We hypothesised that deleting the cytoskeleton binding domain would allow NSP1 to translocate to the nucleus allowing phosphorylated STAT1 and STAT2 to translocate to also the nucleus. SA11-L2 with GS5(NSP1) lacking the cytoskeleton domain was successfully rescued using the SA11-L2 reverse genetics system. Plaque assays and replication kinetics showed that rSA11_NSP1-tr formed smaller plaques than rSA11-L2, but they replicated at a similar rate. These results showed that there could be a higher interferon response in MA104 cells infected with rSA11_NSP1-tr which prevents effective cell-to-cell spread. Further investigations need to be done to determine the localisation of NSP1 lacking the cytoskeleton binding domain and phosphorylated STAT1 and STAT2 in infected cells. This is the first report of the rescue of SA11-L2 with OSU-a NSP4, SA11-L2 with SA11-L2 NSP4 containing aa 135-141 of OSU-a NSP4 and SA11-L2 with NSP1 lacking a cytoskeleton binding domain. These study results show that rationally altering NSP1 and NSP4 could lead to the rescue of viruses that lack some of their pathogenic qualities but can replicate well. Further studies need to be conducted in animal models to evaluate if the rescued viruses do not cause diarrhoea and if there is a greater innate immune response during infection with recombinant rotaviruses. These experiments would be proof of principle that these alterations can increase the safety profile of vaccine candidates.