A SARS-CoV-2 oral vaccine development strategy based on the attenuated Salmonella type III secretion system

Abstract

Aims This study aimed to provide a safe, stable and efficient SARS‐CoV‐2 oral vaccine development strategy based on the type III secretion system of attenuated Salmonella and a reference for the development of a SARS‐CoV‐2 vaccine. Methods and Results The attenuated Salmonella mutant Δ htrA ‐VNP was used as a vector to secrete the antigen SARS‐CoV‐2 based on the type III secretion system (T3SS). The Salmonella pathogenicity island 2 (SPI‐2)‐encoded T3SS promoter (sifB) was screened to express heterologous antigens (RBD, NTD, S2), and the SPI‐2‐encoded secretion system (sseJ) was employed to secrete this molecule (psifB‐sseJ‐antigen, abbreviated BJ‐antigen). Both immunoblotting and fluorescence microscopy revealed effective expression and secretion of the antigen into the cytosol of macrophages in vitro. The mixture of the three strains (BJ‐RBD/NTD/S2, named AisVax) elicited a marked increase in the induction of IgA or IgG S‐protein Abs after oral gavage, intraperitoneal and subcutaneous administration. Flow cytometric analysis proved that AisVax caused T‐cell activation, as shown by a significant increase in CD44 and CD69 expression. Significant production of IgA or IgG N‐protein Abs was also detected by using psifB‐sseJ‐N(FL), indicating the universality of this strategy. Conclusions Delivery of multiple SARS‐CoV‐2 antigens using the type III secretion system of attenuated Salmonella Δ htrA ‐VNP is a potential COVID‐19 vaccine strategy. Significance and Impact of the Study The attenuated Salmonella strain Δ htrA ‐VNP showed excellent performance as a vaccine vector. The Salmonella SPI‐2‐encoded T3SS showed highly efficient delivery of SARS‐COV‐2 antigens. Anti‐loss elements integrated into the plasmid stabilized the phenotype of the vaccine strain. Mixed administration of antigen‐expressing strains improved antibody induction.

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“…The promoter sifB (pSifB) is a Salmonella -specific regulatory expression promoter that is initiated by the intracellular microenvironment, such as low pH and low phosphate. 33 , 34 Fluorescence images showed that the VNP-pSifB-RFP strain selectively expressed RFP after entering macrophages but not in regular media. VNP-pSifB-RFP specifically expressed RFP after entering macrophages, while the strain cultured in normal medium did not (Fig.…”

“…The VNP strain constitutively expressing RFP (VNP-RFP strain), green fluorescent protein (GFP) (VNP-GFP strain), bioluminescence (LuxCDABE) (VNP-Lux strain), and the VNP strain controllably expressing RFP with the aid of the sifB promoter (VNP-psifB-RFP strain) were all constructed as described previously. 33 All plasmids used in this study were constructed using the ClonExpress Ultra One Step Cloning Kit V2 (Vazyme, C116, Nanjing, China). The constructed plasmids, including pJ23100-RFP, pJ23100-Lux, and psifB-RFP, were electrotransformed into VNP competent cells, with the transferred empty vector plasmids serving as blank control strains and denoted as VNP-NC.…”

“…The Salmonella -specific type III secretion system enables specific induction of target protein expression only in macrophages, and we previously screened for potent promoters (sifB) under this system. 33 The strains were monitored in macrophages using Salmonella -specific antibodies (Targetpharma, Nanjing, China). Fluorescence microscopy (Carl Zeiss) proved that intracellularly active bacteria expressed RFP.…”