The role of SARS-CoV-2 nucleocapsid protein in antiviral immunity and vaccine development

Figures & data

Figure 1. The structure of SARS-CoV-2 N-protein. (A). Model of SARS-CoV-2 N-protein dimer. (B). Topology diagram for SARS-CoV-2 N-protein N-terminal RNA-binding domain. (C). Superimposed structures in ribbon representation of N-protein N-terminal RNA-binding domain from SARS-CoV-2 (green), SARS-CoV (cyan), HCoV-OC43 (pink), MERS-CoV (orange), and HCoV-NL63 (blue) [6]. The loops that show significant differences are highlighted by dotted circles. The AMP ligand in the HCoV-OC43 N-protein N-terminal RNA-binding domain-AMP complex is shown as a stick structure. D. Topology diagram for SARS-CoV-2 N-protein C-terminal dimerization domain. E. Superimposed structures in ribbon representation of N-protein C-terminal dimerization domain from SARS-CoV-2 (pink), SARS-CoV (cyan), MERS-CoV (orange) and HCoV-NL63 (blue). Figure adapted from [6].

Figure 2. Induced inflammation by SARS-CoV-2 N-protein.

Table 1. Effects of different human-infecting coronavirus N-proteins on host cell signaling.

Human-infecting coronavirus	Intracellular N-protein											Extracellular N-protein
	Interaction w/ NLRP3	Activation of NF-κB	Interaction w/ Smad3	Interaction w/ GSDMD	Interaction w/ PDK1	α-Synuclein aggregation	Interaction w/ Rig-I	Interaction w/ G3BP1	Interaction w/ MAVS	Interaction w/ STAT1/2	Inhibition of RNA interference	Activation of complement	Activation of NF-κB and MAPK
HCoV-OC43	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
HCoV-NL63	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
HCoV-HKU1	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	× [24]
HCoV-229E	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	× [22]	N/A
MERS-CoV	N/A	N/A	N/A	N/A	N/A	N/A	√ [29–31]	× [32]	N/A	N/A	√ [33]	√ [22]	× [24]
SARS-CoV	N/A	√ [17]	√ [18]	N/A	N/A	N/A	√ [29–31]	√ [32]	N/A	× [34]	√ [33]	√ [22]	× [24]
SARS-CoV-2	√ [15]	√ [16]	√ [17,35]	√ [36]	√ [37]	√ [38]	√ [29–31]	√ [32,39]	√ [40]	√ [30,41]	√ [42,43]	√ [22,23]	√ [24,25]

Note: w/, with; N/A, non-available.

Figure 3. Modulation of cell death by SARS-CoV-2 N-protein.

Table 2. SARS-CoV-2-N-based vaccines under development.

	Vaccine name	N-protein usage	Adverse effects in clinical studies	Protective effects in preclinical studies	Antibody responses	T cell responses	References
1	COH04S1 (a synthetic multiantigen MVA-based vaccine)	Full length N-protein from SARS-CoV-2 ancestral virus, with co-expression of S protein.	Grade 3 fever was observed in one participant who received intramuscular injection of low-dose COH04S1 and placebo, grade 2 anxiety or fatigue was seen in one participant who received medium-dose COH04S1, no severe adverse events were reported in an open-label and randomized, phase 1 trial.	Intramuscular and intranasal vaccination protected Syrian hamsters from weight loss, low respiratory tract infection, and lung pathology following challenge with SARS-CoV-2 ancestral virus and Beta and Delta variants. 2 doses or 1 dose intramuscular vaccination protected African green monkey NHP from lower and upper respiratory tract infection following challenge with SARS-CoV-2 ancestral virus.	Both preclinical and clinical studies: robust binding antibodies to N-protein, robust but rapidly declined virus-neutralizing antibodies which showed reduced cross-reactivity.	Preclinical studies: robust mucosal and systemic IFNγ- and IL-2-expressing N-protein-specific T cells with no or marginally detectable IL-4-expressing N-protein-specific T cells. Clinical studies: blood N-specific T cells elicited in COH04S1-vaccinees demonstrated potent and equivalent cross-reactivity against ancestral SARS-CoV-2 and Delta and Omicron variants.	[69–72]
2	N/A (a vaccine with the LVS ΔcapB vector, a highly attenuated replicating intracellular bacterium)	Full length N-protein from SARS-CoV-2 ancestral virus, with co-expression of M protein.	N/A	Intradermal and intranasal vaccination protected Syrian hamsters from weight loss, low respiratory tract infection, and lung pathology following challenge with SARS-CoV-2 ancestral virus.	Preclinical study: protection correlated with binding antibodies to N-protein.	N/A	[73]
3	N/A (a replication-deficient human adenovirus type 5 vector vaccine)	Full length N-protein from SARS-CoV-2 ancestral virus.	N/A	Intravenous vaccination of Syrian hamsters and K18-hACE2 transgenic C57 BL/6 mice conferred protection, as defined by reduced weight loss and viral load, following challenge with SARS-CoV-2 ancestral virus.	N/A	Preclinical study: rapid and persistent N-specific CD8^+ T cell responses in the respiratory mucosa.	[74]
4	GX-19N (a recombinant DNA vaccine)	Full length N-protein from SARS-CoV-2 ancestral virus, with co-expression of S protein.	The vaccines were delivered intramuscularly using an electroporator. All solicited adverse events were mild; no serious vaccine-related adverse events were detected.	N/A	Clinical study: no information about binding antibodies to N-protein, virus-neutralizing antibodies were weaker than those induced by commercial vaccines.	Clinical study: broad N-protein-specific T-cell responses in the blood.	[75]
5	SpiN (a fusion protein vaccine)	Full length N-protein from SARS-CoV-2 ancestral virus, in fusion with RBD of S protein.	N/A	Intramuscular immunization of Syrian hamsters and K18-ACE2 transgenic C57 BL/6 mice with poly I:C as the adjuvant promoted robust resistance to SARS-CoV-2 ancestral virus, as indicated by viral load, lung inflammation, weight loss, and reduction of lethality. This vaccine also protected K18-ACE2 transgenic C57 BL/6 mice against infection with Delta and Omicron variants.	Preclinical study: robust binding antibodies to N-protein, no virus-neutralizing antibodies.	Preclinical study: robust mucosal and systemic IFNγ response by N-protein-specific T cells.	[76]
6	Tri:HuAd (a replication-deficient human adenovirus type 5 vector vaccine) or Tri:ChAd (a replication-deficient chimpanzee adenovirus type 68 vector vaccine)	Full length N-protein from SARS-CoV-2 ancestral virus, in fusion with a highly conserved region of RNA-dependent RNA polymerase and with co-expression of S1 domain of S protein.	N/A	Intranasal, but not intramuscular, immunization of BALB/c mice provided potent B- and T-cell-dependent protection from the lethal dose infection with mouse-adapted strain of SARS-CoV-2 ancestral virus. Single-dose intranasal immunization with ChAd-vectored trivalent COVID-19 vaccine protected against lethal infection by SARS-CoV-2 variants of concern.	Preclinical study: no information about binding antibodies to N-protein, robust virus-neutralizing antibodies.	Preclinical study: intranasal vaccination induced robust mucosal and systemic IFNγ-, TNFα-, or IL-2-expressing monofunctional N-protein-specific CD8^+ T cells, robust mucosal and systemic IFNγ-expressing N-protein-specific CD4^+ T cells, and respiratory mucosal tissue-resident memory T cell responses.	[77]
7	N/A (a replication-deficient human adenovirus type 5 vector vaccine)	Full length N-protein from SARS-CoV-2 ancestral virus, with co-expression of S protein.	N/A	Intramuscular vaccination of Syrian hamsters and K18-hACE2 transgenic C57 BL/6 mice conferred acute protection in both the lung and brain following challenge with SARS-CoV-2 ancestral virus.	Preclinical study: robust binding antibodies to N-protein, no information about virus-neutralizing antibodies.	Preclinical study: robust local (lung and brain) and systemic IFNγ-expressing N-protein-specific CD8^+ T cells, robust systemic IFNγ-expressing N-protein-specific CD4^+ T cells.	[78]
8	N/A (a recombinant protein vaccine with flagellin and cyclic GMP-AMP as adjuvants)	Full length N-protein from SARS-CoV-2 ancestral virus, with co-expression of prefusion-full S protein	N/A	Intranasal vaccination of K18-hACE2 transgenic C57 BL/6 mice protected against lethal SARS-CoV-2 ancestral virus challenge, with superior protection in the upper respiratory tract compared with that in mice immunized with an inactivated vaccine.	Preclinical study: robust binding antibodies to N-protein, robust IgA^+ B cells, robust virus-neutralizing antibodies which reduced cross-reactivity.	Preclinical study: robust mucosal and systemic IFNγ- or IL-4-expressing N-protein-specific T cells with equivalent cross-reactivity against ancestral SARS-CoV-2 and Beta and Delta variants, more mucosal CD8^+ T cells, respiratory mucosal tissue-resident CD4^+ and CD8^+ T cells.	[79]
9	UB-612 (a multitope subunit vaccine)	Rationally designed promiscuous peptides representing sarbecovirus conserved CD4^+ ad CD8^+ T cell epitopes on N, M, and S2 proteins, and S1-RBD-sFc protein.	No vaccine-related serious adverse events were recorded after intramuscular vaccination. The most common solicited adverse events were injection site pain and fatigue, mostly mild and transient.	N/A	Clinical studies: no information about N-protein-specific antibodies, potent neutralizing titers against ancestral SARS-CoV-2 virus, Delta, Omicron, and other variants of concern, but the virus-neutralizing antibodies were long-lasting as revealed with the live ancestral SARS-CoV-2 virus.	Clinical studies: no information about N-protein-specific T cells, restimulation of peripheral blood mononuclear cells with designer peptide antigens revealed long-lasting robust Th1-predominant cell response as measured by IFN-γ and IL-4 ELISpot.	[80]
10	N/A (a nucleoside-modified mRNA vaccine)	A mRNA vaccine expressing fulll length N-protein from SARS-CoV-2 ancestral virus, alone or in combination with the clinically proven S-expressing mRNA vaccine.	N/A	Intramuscular, but not intranasal, immunization of BALB/c mice and Syrian hamsters with N-protein mRNA induced modest control of mouse-adapted SARS-CoV-2, as indicated by viral load and body weight. In BALB/c mice, dual S- and N-protein mRNA vaccination further lowered viral load. In Syrian hamsters, dual S- and N-protein mRNA vaccination not only induced more robust control of the Delta and Omicron variants in the lungs but also provided enhanced protection in the upper respiratory tract.	Preclinical study: intramuscular vaccination with N-protein mRNA alone induced robust binding antibodies to N-protein but no neutralizing activity, dual S- and N-protein mRNA vaccination augmented serum neutralizing antibody activities.	Preclinical study: intramuscular vaccination with N-protein mRNA alone induced robust systemic N-protein-specific CD4^+ and CD8^+ T cell responses for IFNγ-, TNFα-, and IL-2, dual S- and N-protein mRNA vaccination augmented systemic S-protein-specific CD8^+ T cell response.	[81]

Note: N/A, non-available; NHP, non-human primates.

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