T7 expression plasmids for producing a recombinant human G1P[8] rotavirus comprising RIX4414 sequences of the RV1 (Rotarix , GSK) vaccine strain

ABSTRACT The live oral rotavirus RV1 (Rotarix) vaccine is formulated from the human G1P[8] RIX4414 virus. Based on RIX4414 sequences, T7 expression plasmids were constructed that supported recovery of recombinant RIX4414-like viruses by reverse genetics. These plasmids will advance the study of the RV1 vaccine, possibly allowing improvements to its efficacy.

R V1 (Rotarix , GSK), the most widely used rotavirus vaccine, is formulated from the human G1P [8] virus RIX4414 (1,2).To protect against rotavirus gastroenteri tis, >24 million children received the RV1 vaccine in 2021 (2).A challenge to rotavirus immunization efforts is that RV1 and other rotavirus vaccines have vaccine efficacies in low-income countries (50%-64%) that can be significantly lower than in high-and moderate-income countries (85%-98%) (3,4).We report here the construction of T7 plasmids that allow for the recovery of a recombinant (r) RIX4414-like virus by reverse genetics.By this method, it may be possible to generate modified forms of the RV1 vaccine with improved performance in low-income countries.
The rotavirus genome consists of 11 segments of double-stranded RNA (5).The rotavirus strain RIX4414 (originally named 89-12) was isolated from a child with acute gastroenteritis in 1989 and serially passaged in cell culture to promote the introduction of attenuating mutations (6).In this study, we designed 11 pT7 expression plasmids, each containing a cDNA sequence corresponding to one of the RIX4414 genome segments, using sequencing information for RIX4414 available in GenBank (Table 1).In cases where RIX4414 sequence information was missing, vis-a-vis, portions of the 5' and 3'-untransla ted regions, sequence information for the prototypic human G1P [8] Wa virus was used instead (Table 1).Because the original pT7/RIX4414 VP2 plasmid was not functional in the reverse genetics system, the VP2 coding region was slightly modified to include residues common to other human G1/4P [8] virus strains [e.g., Wa, KU (7), Odelia ( 8)] (Table 1).To generate pT7/RIX4414 plasmids, RIX4414 cDNA sequences were made with an upstream T7 promoter and downstream hepatitis D virus ribozyme and T7 terminator, by Gibson assembly of oligonucleotide fragments (9,10).These cDNAs were inserted into the EcoRV site of pUC-GW-Amp plasmids (Azenta).
The pT7/RIX4414 plasmids supported recovery of rRIX4414-like virus using a modified reverse genetics procedure (11,12).Briefly, BHK-T7 monolayers in 12-well plates were transfected with plasmid mixtures containing 0.8 µg of each pT7/RIX4414 plasmid (except the NSP2 and NSP5 plasmids, which were 4.8 µg each), 1.6 µg of pCMV/NP868R capping plasmid, and 1.6 µg of pcDNA/T7 RNA polymerase plasmid.Two days later, the transfected cells were overseeded with 10 5 MA104 cells.Three days later, the BHK-T7/ Full, T7 expression plasmid contains a complete cDNA sequence for a RIX4414 genome segment matching the GenBank number.Partial, complete rotavirus cDNA sequence in T7 expression plasmid was generated by combining the partial RIX4414 segment sequence reported by the GenBank number with the 5' and/or 3' UTR sequences of other Wa-like rotaviruses.
MA104 cells was overseeded with 10 5 Vero cells.Eight days later, viruses in the cell lysates were amplified on Vero cells and plaque isolated on MA104 cells (13).The RNA genome of the rRIX4414-like isolate is shown in Fig. 1, and the genome sequence of the rRIX4414like isolate was confirmed by Nanopore sequencing (14).Based on sequence analysis, the rRIX4414-like virus has nucleotide and amino acid sequence identities of >99% with the vaccine RIX4414 (lab-strain) virus (accession numbers for the lab strain provided in Table 1), making the rRIX4414 reverse genetics system a possible tool for investigating genetic changes that may improve RV1 performance.

TABLE 1
RIX4414 sequences used in the generation of T7 expression plasmids