Engineering rotavirus-based vaccine vectors expressing SARS-CoV-2 spike epitopes

In a recent study posted to the bioRxiv* preprint server, researchers proposed rotavirus (RV)-based vaccine vectors with the potential for developing polyvalent vaccines targeting multiple enteric viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19).

Background

Previously, the researchers used live attenuated platforms to manufacture RV-based vaccines. However, a SARS-CoV-2 peptide insertion as small as 34 amino acids (AA) into the hypervariable region of the viral protein 4 (VP4) of RV from the SA11 strain resulted in reduced viral titer and replication, thus limiting its use as a potential vaccine expression platform.

Contrastingly, recombinant RVs harboring chimeric VP4 proteins have shown efficient replication in cell culture and neutralizing activity in mice. Although previous studies have not tested peptide insertions into VP4, it's potential to express heterologous epitopes from different RV strains makes it an appealing delivery platform for several vaccines.

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About the study

In the present study, researchers used a plasmid-based reverse genetics system for RVs to test the tolerance of RVs for peptide insertions and conceptualize novel vaccine platforms.

To this end, they engineered SA11 strain VP4 plasmids with SARS-CoV-2 spike (S) peptide sequences inserted into the hypervariable region; likewise, they synthesized a panel of bovine RF strain RV non-structural protein 3 (NSP3) plasmids. They tagged the C-terminal of the NSP3 open reading frame (ORF) with 193 AA long receptor-binding domain (RBD), or the relatively shorter receptor binding motif (RBM) located within the RBD of the SARS-CoV-2 S. They performed peptide insertions with or without translational self-cleaving 2A (T2A)-separation.

Notably, the RBD of the SARS-CoV-2 S is the most immunogenic antigen. Thus, inserting RBD peptides into the RV backbone helped the researchers determine whether chimeric RVs showed cross-reactivity against SARS-CoV-2 S antibodies.

The team generated mutant viruses, including VP4 mutants and NSP3 mutants, by replacing plasmids encoding the SA11 VP4 or RF NSP3 gene segments with corresponding plasmids encoding SARS-CoV-2 S epitopes. They performed mutant rescue experiments three times for each virus panel and titered these using plaque assays. Lastly, they confirmed the presence of mutations in the target gene segments using reverse transcription-polymerase chain reaction (RT-PCR).

Study findings

The mutated hypervariable region of VP4 of the simian SA11 RV strain hampered viral replication; however, T2A-separation did not compromise viral replication. Consequently, tagged NSP3 elicited cross-reactivity with SARS-CoV-2 S antibodies, as observed under enzyme-linked immunosorbent assay (ELISA).

On the contrary, ELISA did not detect any cross-reactivity of the SARS-CoV-2 S peptides with S or RBD antibodies, suggesting the inefficiency of the SA11 VP4 mutant as an expression vector for foreign peptides. However, the VP4 mutants were detected in the Western blot test, raising the possibility of the immunogenicity of other peptides when presented in this way.

Nevertheless, the study demonstrated that bovine RF strain-derived NSP3 could tolerate insertions of foreign peptide sequences at its C-terminus. However, in the absence of the T2A element, there may be an upper limit to the number of foreign peptide sequences that the RF NSP3 could accommodate.

Introducing SARS-CoV-2 S peptides into the viral protein 8 (VP8) of the VP4 mutant between amino acid positions 164 – 198 showed a substantial decrease in viral titers without affecting the mutant rescue efficiency.

The researchers rescued an RF mutant having NSP3 directly fused with 193 AA long RBD peptide only once (1/3), suggesting an impaired NSP3 function. They also observed that the RNA: plaque-forming units (PFU) ratio was affected in the RBD mutant only; likewise, copy numbers of NSP3/VP1 encoding transcripts were affected too. These findings also suggest that NSP3 plays a crucial role in virus replication by regulating viral messenger ribonucleic acid (mRNA) translation; moreover, the observed increase in RNA: PFU ratios was not specific to a particular peptide segment.

Conclusions

The study demonstrated the possibility of developing recombinant vaccines utilizing a bovine RF strain RV as a backbone.

Since the VP4 mutants tagged with smaller peptides from SARS-CoV-2 S showed diminished infectivity, they may not serve as a potential vaccine expression platform. On the contrary, all the NSP3 mutants expressing SARS-CoV-2 S peptides, except T2A_RBM, accommodated relatively large foreign peptide sequences. Also, they cross-reacted with the SARS-CoV-2 RBD antibody, suggesting the viability of the NSP3 as a tagging system and demonstrating its application to other RV strains.

In the future, follow-up studies must be conducted using sera from COVID-19 patients to confirm antibody responses to the antigens produced by the NSP3 mutants.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.