Reverse Genetics Approach for Developing Rotavirus Vaccine Candidates Carrying VP4 and VP7 Genes Cloned from Clinical Isolates of Human Rotavirus

Abstract

Species A rotaviruses (RVs) are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years. Currently available RV vaccines were adapted from wild-type RV strains by serial passage of cultured cells or by reassortment between human and animal RV strains. These traditional methods require large-scale screening and genotyping to obtain vaccine candidates. Reverse genetics is a tractable, rapid, and reproducible approach to generating recombinant RV vaccine candidates carrying any VP4 and VP7 genes that provide selected antigenicity. Here, we developed a vaccine platform by generating recombinant RVs carrying VP4 (P[4] and P[8]), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P[2]) as a backbone. Neutralization assays using monoclonal antibodies and murine antisera revealed that recombinant VP4 and VP7 monoreassortant viruses exhibited altered antigenicity. However, replication of VP4 monoreassortant viruses was severely impaired. Generation of recombinant RVs harboring a chimeric VP4 protein for SA11 and human RV gene components revealed that the VP8* fragment was responsible for efficient infectivity of recombinant RVs. Although this system must be improved because the yield of vaccine viruses directly affects vaccine manufacturing costs, reverse genetics requires less time than traditional methods and enables rapid production of safe and effective vaccine candidates.IMPORTANCE Although vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral antigenicity, growth capacity, and pathogenicity. Here, we generated recombinant simian RVs (strain SA11) carrying heterologous VP4 and VP7 genes cloned from clinical isolates and showed that VP4- or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.

Keywords: reverse genetics; rotavirus; vaccine.

FIG 1

Preparation of hVP4 and hVP7 genes from human RV clinical isolates. (A and B) Phylogenetic analysis of VP4 and VP7 genes cloned from human RV strains. The deduced amino acid sequences of VP4 (A) and VP7 (B) genes cloned from human RV clinical isolates (black circles) were analyzed together with RV reference strains. Phylogenetic trees were constructed by the neighbor-joining method. The SA11 strain used as the backbone virus is indicated by a white circle. Scale bars denote the number of nucleotide substitutions per site. For further phylogenetic analyses of G3 RV strains see Fig. 2 (C to E) Isolation of human RV clinical samples. (C) Viral dsRNA profile of human RV isolates. (D) Detection of RV antigens in cells infected with human RVs using anti-SA11 serum and an anti-SA11 NSP4 antibody. (E) Replication kinetics of human RVs in MA104 cells. The growth kinetics of rSA11 are shown for comparison.

FIG 2

Phylogenetic tree of G3 VP7 genes. The nucleotide sequences of G3 VP7 genes of human and equine RVs deposited in GenBank were aligned and a phylogenetic tree was constructed using the maximum likelihood method. Bootstrap values higher than 60% are shown. RVA-U4 (black circle) and SA11 (white circle) are indicated. The scale bar denotes the number of nucleotide substitutions per site.

FIG 3

Generation of recombinant simian RVs carrying VP4 and VP7 genes cloned from human RV clinical isolates. (A and B) Electropherotypes of dsRNA genomes purified from rSA11-hVP4 (A) and rSA11-hVP7 (B) viruses. Bands corresponding to VP4 (A) and VP7 (B) genes are indicated by arrowheads. (C and D) Replication of rSA11-hVP4 (C) and rSA11-hVP7 (D) monoreassortant viruses. MA104 cells were infected with rSA11-hVP4 and rSA11-hVP7 viruses at an MOI of 0.0001 FFU/cells and incubated for various durations. Virus infectious titers in the cell lysates were determined by a focus-forming assay.

FIG 4

Contributions of the VP4, VP6, and VP7 genes to viral replication. (A and B) Generation of mono and double reassortant viruses carrying the hVP4, hVP6, and/or hVP7 genes in the SA11 backbone. (A) Electropherotypes of dsRNA genomes purified from rSA11 carrying the hVP4, hVP6, and/or hVP7 genes. (Left) The combinations of human/simian VP4, VP6, and VP7 genes in each recombinant virus are indicated. S, SA11; H, human clinical isolate (RVA-U14). (Right). RNA migration patterns of the mono and double reassortant viruses. (B) Replication of rSA11-hVP4, -hVP6, and -hVP7 reassortant viruses. MA104 cells were infected with reassortant viruses at an MOI of 0.0001 FFU/cells. At 48 h postinfection, virus infectious titers in the cell lysates were determined by a focus-forming assay. (C and D) Generation of recombinant human RV carrying the VP4 gene of the SA11 strain. MA104 cells were infected with chimeric VP4 reassortant viruses at an MOI of 0.0001 FFU/cells. At 48 h postinfection, virus infectious titers in the cell lysates were determined by a focus-forming assay. (E) Generation of chimeric VP4 monoreassortant viruses between simian RV (SA11) and human RV (RVA-U14). (Left) Construction of various chimeric VP4 monoreassortant viruses between the SA11 strain and RVA-U14. Light blue indicates the SA11 VP4 gene sequence. Light salmon indicates the human RV VP4 gene sequence. Numbers indicate nucleotide length. (Right) MA104 cells were infected with chimeric VP4 reassortant viruses at an MOI of 0.0001 FFU/cells. At 48 h postinfection, virus infectious titers in the cell lysates were determined by a focus-forming assay.

FIG 5

Examination of the antigenicity of hVP4 and hVP7 reassortant viruses using neutralizing monoclonal antibodies against SA11 VP4. (A to D) Epitope mapping of the MAb-68 recognition site. (A) Crystal structure of the trimer of VP4 spike proteins in infectious virus particles (RV strain RRV, PDB 3IYU). The three VP4 proteins in the trimer are denoted by pink, light green, and light blue. VP7 trimers forming the virion surface are colored gray. Aspartate (D) residues at position 358 are shown as red spheres, and arginine residues at position 441 are shown as blue spheres. (B) Reactivity of MAb-68 against recombinant VP4 protein. Hemagglutinin (HA) peptide-tagged SA11 VP4 or VP4-D358N proteins expressed in MA104 cells were detected by labeling with MAb-68 and rabbit anti-HA antibodies, followed by anti-mouse IgG CF594 and anti-rabbit IgG CF488 conjugates, respectively. (C) Neutralization assay of MAb-68 against rSA11 and rSA11-VP4-D358N. (D) Partial amino acid sequences of the VP5* fragment of SA11 and human RV strains. The assumed recognition sites of MAb-29 and MAb-68 are indicated. (E and F) Neutralization assay of MAb-29 and MAb-68 against rSA11-hVP4 and rSA11-hVP7 monoreassortant viruses. Viruses were incubated with MAb-29 (E) or MAb-68 (F) at 37°C for 1 h. MA104 cells were inoculated with the mixture of virus and MAb, and virus-infected cells were detected by immunofluorescence. The reciprocal value of the serum dilution that yielded 50% inhibition was calculated as the neutralization titer.

FIG 6

Examination of the antigenicity of hVP4 and hVP7 monoreassortant viruses using murine antisera obtained by immunization with recombinant RVs. (A) Neutralization activities of murine antisera raised by repeated infection of rSA11 (n = 6), rSA11-hVP4/U3_P[8] (n = 5), and rSA11-hVP7/U8_G9 (n = 5) against homologous viruses were examined. (B to D) Heterologous neutralization activities of murine antisera generated by immunization with rSA11 (B), rSA11-hVP4/U3_P[8] (C), and rSA11-hVP7/U8_G9 (D). The relative heterologous neutralization activities in comparison to the homologous neutralization activities were calculated. The deduced serotypes of each virus are indicated after the virus IDs. Arrows indicate neutralization activities against homologous viruses.