Enhanced immunogenicity elicited by a novel DNA vaccine encoding the SARS-CoV-2 S1 protein fused to the optimized flagellin of Salmonella typhimurium in mice.

Abstract

The development of safe and efficient vaccines against coronavirus disease 2019 (COVID-19) is necessary for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccines. Among the available vaccine types, DNA vaccines are a promising alternative to conventional vaccines. Here, we developed a novel DNA vaccine containing the SARS-CoV-2 S1 subunit and an optimized flagellin adjuvant (FliCΔD2D3) from Salmonella typhimurium. The S1 gene and S1-fliCΔD2D3 fusion gene were codon optimized and then cloned into the antibiotic-resistance-gene-free vector asd-pVAX1, producing asd-pVAX1-S1 and asd-pVAX1-S1-fliCΔD2D3, respectively. The expression of S1 and S1-FliCΔD2D3 proteins was confirmed after transient expression in HEK293T cells. After in vitro characterization of the vaccine candidates, we evaluated the humoral and cellular immune responses induced in BALB/c mice. The DNA vaccine induced a long-lasting humoral response for approximately 4 months, and the elicited serum antibodies efficiently blocked the binding of the SARS-CoV-2 S1 receptor-binding domain to its entry receptor (angiotensin-converting enzyme 2). Moreover, the DNA vaccine induced strong Th1/Th2-mixed immune responses, as demonstrated by the similar levels of S1-specific immunoglobulin (Ig) G1 and IgG2a in the serum and the significantly elevated Th1-type (IFN-γ and TNF-α) and Th2-type (IL-4 and IL-6) cytokine expression from splenic lymphocytes. Importantly, asd-pVAX1-S1-fliCΔD2D3 immunization in mice could significantly enhance the levels of immune responses compared to asd-pVAX1-S1. This study provides crucial information regarding the selection of a safer DNA vector and a new adjuvant for the vaccine development of SARS-CoV-2 and other infectious diseases. IMPORTANCE The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.