Abstract
Among vaccines administered to children are those targeting rotavirus, a segmented double-stranded RNA virus that represents a major cause of severe gastroenteritis. To explore the feasibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding cassettes for NSP3, a translational 2A stop-restart signal, and a FLAG-tagged portion of the SARS-CoV-2 spike (S) protein: S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended RBD (ExRBD), or S2 core (CR) domain. Generation of rSA11 containing the S1 coding sequence required a sequence insertion of 2.2 kbp, the largest such insertion yet introduced into the rotavirus genome. Immunoblotting showed that rSA11 viruses containing the smaller NTD, RBD, ExRBD, and CR coding sequences expressed S-protein products of expected size, with ExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, the rSA11 virus containing the full-length S coding sequence (rSA11/NSP3-fS1) failed to express its expected 80 kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of immunogenic portions of the SARS-CoV-2 S protein, including NTD, RBD, ExRBD, and CR, that have sizes smaller than the S1 fragment.
Highlights
The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on human mortality and morbidity has stimulated-broad ranging efforts to develop vaccines preventing coronavirus disease 19 (COVID-19) [1,2]
To examine the possibility of using rotavirus as an expression platform for regions of the SARS-CoV-2 S protein, we replaced the NSP3 open-reading frame (ORF) in the pT7/NSP3SA11 transcription vector with a cassette comprised of the NSP3 ORF, a porcine teschovirus 2A element, and a coding sequence of the S protein (Figure 2)
The cassette included a flexible GAG hinge between the coding sequence for NSP3 and the 2A element and a 3x FLAG (f) tag between the coding sequences for the 2A element and the S region. This approach was used to generate a set of vectors that contained coding sequences for SARS-CoV-2 S1, N-terminal domain (NTD), receptor-binding domain (RBD), an extended form of the RBD (ExRBD), and the S2 core region (CR) including its fusion domains (Figure 1)
Summary
The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on human mortality and morbidity has stimulated-broad ranging efforts to develop vaccines preventing coronavirus disease 19 (COVID-19) [1,2]. The most widely used rotavirus vaccines are given orally and formulated from live-attenuated virus strains [8]. These vaccines induce the production of neutralizing IgG and IgA antibodies [9,10,11] and have been highly effective in reducing the incidence of rotavirus hospitalizations and mortality [12,13]. Rotavirus reverse genetics systems have been used to mutate several of the viral genome segments and to generate virus strains that express reporter proteins [17,19,26,27,28,29,30]
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