Recombinant COVID-19 vaccine based on recombinant RBD/Nucleoprotein and saponin adjuvant induces long-lasting neutralizing antibodies and cellular immunity

Abstract

SARS-CoV-2 has caused a global pandemic, infecting millions of people. An effective preventive vaccine against this virus is urgently needed. Here, we designed and developed a novel formulated recombinant receptor-binding domain (RBD) nucleocapsid (N) recombinant vaccine candidates. The RBD and N were separately expressed in E. coli and purified using column chromatography. The female Balb/c mice were immunized subcutaneously with the combination of purified RBD and N alone or formulated with saponin adjuvant in a two-week interval in three doses. Neutralization antibody (Nabs) titers against the SARS-CoV-2 were detected by a Surrogate Virus Neutralization (sVNT) Test. Also, total IgG and IgG1, and IgG2a isotypes and the balance of cytokines in the spleen (IFN-γ, Granzyme B, IL-4, and IL-12) were measured by ELISA. The percentages of CD4+ and CD8+ T cells were quantified by flow cytometry. The lymphoproliferative activity of restimulated spleen cells was also determined. The findings showed that the combination of RBD and N proteins formulated with saponin significantly promoted specific total IgG and neutralization antibodies, elicited robust specific lymphoproliferative and T cell response responses. Moreover, marked increase in CD4+ and CD8+ T cells were observed in the adjuvanted RBD and N vaccine group compared with other groups. The results suggest that the formulations are able to elicit a specific long-lasting mixed Th1/Th2 balanced immune response. Our data indicate the significance of the saponin-adjuvanted RBD/N vaccine in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective long-lasting and strong vaccine.

Saponins: Research Progress and Their Potential Role in the Post-COVID-19 Pandemic Era

“…In this context, it has been demonstrated that the protective immunity of certain types of vaccines could be enhanced by the addition or substitution of different adjuvants [ 120 , 121 ]. Between these adjuvants, saponins are one of the most widely used immunostimulants to boost vaccines because they may modify the activities of T cells and activate anti-gen-presenting cells (APCs), such as dendritic cells (DC), and induce Th1/Th2 adaptive immunoresponses (Th1 or cell-mediated immunity controlled by activated T cells, and Th2 or humoral immunity controlled by activated B cells and antibodies) or only Th2 response [ 36 , 49 , 120 , 121 , 122 , 123 , 124 , 125 ].…”

“…Between these, adjuvant systems based on QS-21 have been widely suggested and studied as an alternative for boosting recombinant vaccines. These systems use the complete S protein as an immunogen [ 36 , 49 , 120 ], or its S1 domain [ 150 ] or only its receptor binding domain (RBD)/nucleocapsid (N) [ 122 ]. Currently, these adjuvant systems are being studied in clinical trials (i.e., still in progress), in which it is proposed to evaluate the safety, reactogenicity, and immune response against COVID-19 of a recombinant S protein-based vaccine with the QS-21 adjuvant (NCT04784767).…”

“…However, when the loss of the acyl chain occurs, the fucosyl unit at the reducing end of the C28 oligosaccharide is exposed for its interaction with C-type lectin receptors (CLRs), specifically, with dendritic cell-specific intercellular adhesion molecule3-grabbing non-integrin (DC-SIGN) receptors, skewing the DCs to a single Th2 immunity [ 121 , 123 , 133 ]. It has also been described that the aldehyde group in position C4 of the saponin can form an imine with the ɛ-amino group of the T lymphocyte receptor (most likely CD2) triggering the activation of the MAPK signaling pathway, changes in transport channels of K + and Na + , and finally skewing the activated T cells to a Th1 immunity with increased production of Th1 cytokines [ 49 , 122 , 124 ]. (b) Immunostimulatory mechanism of action for QS-21.…”

Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain (RBD) Produced in E. coli Combined with the Army Liposomal Formulation Containing QS21 (ALFQ) Elicits Neutralizing Antibodies against Mismatched Variants

“…Despite this, refolded RBDs have demonstrated binding to SARS-CoV-2 antibodies [ 49 ] and the human ACE2 receptor [ 31 , 50 ], and their overall structure was similar to mammalian-derived products [ 31 , 53 ]. E. coli RBD vaccination in small animals with multiple adjuvants, including Al(OH) 3 + CpG ODN [ 51 ], Sigma Adjuvant System (Sigma-Aldrich) [ 47 ], TiterMax Gold (Sigma-Aldrich) [ 50 ], Alum [ 28 ], and Saponin [ 54 ], have shown high immunogenicity and the induction of neutralizing antibodies. Soluble proteins that are generally considered weak immunogens require formulation with potent adjuvants [ 55 ].…”

The SARS-CoV-2 nucleocapsid protein: its role in the viral life cycle, structure and functions, and use as a potential target in the development of vaccines and diagnostics

“…Priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective [ 164 ]. Similarly, other teams also designed and developed multiple recombinant vaccines based on nucleoproteins [ 165 – 168 ].The results showed that recombinant vaccines could significantly increase the levels of serum neutralizing antibody and total immunoglobulin [ 165 ], and induce strong specific lymphocyte proliferative response and T cell response [ 165 ]. The antigen specificity level of interferon-γ in splenocytes of immunized mice increased [ 166 , 167 ].…”