Two formulations of coronavirus disease‐19 recombinant subunit vaccine candidate made up of S1 fragment protein P1, S2 fragment protein P2, and nucleocapsid protein elicit strong immunogenicity in mice

Abstract

Introduction: Coronavirus disease (COVID-19) is ongoing as a global epidemic and there is still a need to develop much safer and more effective new vaccines that can also be easily adapted to important variants of the pathogen. In the present study in this direction, we developed a new COVID-19 vaccine, composed of two critical antigenic fragments of the S1 and S2 region of severe acute respiratory syndrome coronavirus 2 as well as the whole nucleocapsid protein (N), which was formulated with either alum or alum plus monophosphoryl lipid A (MPLA) adjuvant combinations. Methods: From within the spike protein S1 region, a fragmented protein P1 (MW:33 kDa) which includes the receptor-binding domain (RBD), another fragment protein P2 (MW:17.6) which contains important antigenic epitopes within the spike protein S2 region, and N protein (MW:46 kDa) were obtained after recombinant expression of the corresponding gene regions in Escherichia coli BL21. For use in immunization studies, three proteins were adsorbed with aluminum hydroxide gel and with the combination of aluminum hydroxide gel plus MPLA.Results: Each of the three protein antigens produced strong reactions in enzyme-linked immunosorbent assays and Western blot analysis studies performed with convalescent COVID-19 patient sera. In mice, these combined protein vaccine candidates elicited high titer anti-P1, anti-P2, and anti-N IgG and IgG2a responses. These also induced highly neutralizing antibodies and elicited significant cell-mediated immunity as demonstrated by enhanced antigen-specific levels of interferon-γ (INF-γ) in the splenocytes of immunized mice.

Overview of Nucleocapsid-Targeting Vaccines against COVID-19

“…A combined vaccine consisting of two immunogenic spike protein regions and a full-length N antigen formulated with aluminum hydroxide with or without monophosphoryl lipid A was studied by Özcengiz et al The authors demonstrated recognition of the recombinant proteins by the sera of COVID-19 convalescents, as well as induction of high titers of anti-P1, anti-P2 and anti-N IgG (including neutralizing antibodies) and interferon-gamma (IFN-γ) secreting T cells in mice. The developers note that the vaccine with adjuvant containing monophosphoryl lipid A is more suitable for immunization of elderly individuals, because it promotes the development of Th1 immune response, while the use of aluminum hydroxide alone is more justified for vaccination of young people [ 51 ].…”

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][166][167][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].…”

“…Similarly, other teams also designed and developed multiple recombinant vaccines based on nucleoproteins [165][166][167][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]. The recently published reviews provided a good summary of the role of SARS-CoV-2 nucleocapsid protein in antiviral immunity and vaccine development [169,170].…”

The Key to Increase Immunogenicity of Next-Generation COVID-19 Vaccines Lies in the Inclusion of the SARS-CoV-2 Nucleocapsid Protein

“…Proteins Induces Immune Response. An intriguing approach, combining the N protein with subunit S1 or S2 of the spike protein utilizing BALB/c mice demonstrated a higher induction of specific S1, S2, and N IgG antibodies and neutralizing activity and IFN-γ production in all immunized animals [80].…”

“…An intriguing approach, combining the N protein with subunit S1 or S2 of the spike protein utilizing BALB/c mice demonstrated a higher induction of specific S1, S2, and N IgG antibodies and neutralizing activity and IFN- γ production in all immunized animals [ 80 ]. Hong et al [ 81 ] investigated the combination of the N protein with the S protein using an E. coli expression system, wherein they fused the RBD of the spike protein with the tetanus toxoid epitope P2 and the whole N protein.…”