Next-Generation Vaccines against COVID-19 Variants: Beyond the Spike Protein

Abstract

Vaccines are among the most effective medical countermeasures against infectious diseases. The emergence of the Coronavirus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred scientific strategies to fight against the disease. Since 2020, in response to the pandemic, many vaccines based on different platforms have been under development, among which mRNA, adenoviral vectors, and subunit vaccines have been clinically approved for use in humans. These first-generation COVID-19 vaccines largely target the viral spike (S) protein and are aimed at eliciting potent neutralizing antibodies. With the emergence of SARS-CoV-2 variants, particularly the highly transmissible Omicron strains, S-based vaccine strategies have faced a continuing challenge of strong immune escape by variants. The coronavirus nucleocapsid (N) protein is a viral protein that induces strong T-cell immunity and is more conserved than S protein across different SARS-CoV-2 variants. Inclusion of N protein in the development of COVID-19 vaccines has been reported. Here, we briefly review and discuss COVID-19, current S-protein-based vaccine strategies, the immunobiology of N protein in SARS-CoV-2 host immunity, and next-generation vaccine strategies involving N protein to combat current and emerging SARS-CoV-2 variants.

Main article text

INTRODUCTION

In early 2020, the causative agent of Coronavirus Disease 2019 (COVID-19) was identified as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [1, 2]. Although the precise origin of the virus is unknown, it shows high similarity with several bat coronaviruses. After the World Health Organization issued global warnings, SARS-CoV-2 crossed international borders and led to a pandemic [3]. As of December 2022, SARS-CoV-2 has caused 650 million infections and 6.6 million deaths globally [4]. Compared with other beta-coronaviruses (e.g., Middle East Respiratory Syndrome Coronavirus [MERS-CoV] and SARS-CoV), SARS-CoV-2 has a higher replication rate and attack rate in communities and induces high fatality rates in hospitalized patients [5]. Nevertheless, the infection and recovery patterns scarcely depend on demographic factors [6], and the underlying mechanisms are not fully understood to date. COVID-19 symptoms are similar to those of influenza, including fever, frontal headache, retro-orbital or temporal headache, gastrointestinal symptoms, ground-glass opacity, pleural effusion [7], loss of taste or smell, and neurological symptoms. Laboratory findings including lymphopenia, hypersensitive C-reactive protein, erythrocyte sedimentation rate, creatine kinase, transaminases (aspartate aminotransferase and alanine aminotransferase), thrombocytopenia are more common biomarkers for diagnosis in patients with COVID-19 [8], and thus may serve as biomarkers [8]. Similarly, lung radiographic findings indicate ground grass opacity, predominantly peripheral distribution, and interlobular septal thickening in patients with COVID-19. Patients with severe COVID-19 and respiratory distress are transferred to intensive care for mechanical ventilation. Of note, SARS-CoV-2 infection severity is associated with comorbidities, such as cardiovascular diseases, diabetes, and obesity, thus leading to high mortality [9, 10].

COVID-19 PATHOPHYSIOLOGY

COVID-19 pathophysiology has been extensively discussed elsewhere [11–13]. Detailed research on host-virus interactions has revealed that SARS-CoV-2 enters hosts through different routes, including nasal and oral routes, and binds the functional receptor angiotensin-converting enzyme 2 (ACE2) [14]. ACE2 is highly expressed on alveolar epithelial cells, enterocytes, endothelial cells, and the oral mucosa. The spike (S) protein of SARS-CoV-2 enters cells via ACE2, and SARS-CoV-2 RNA subsequently is replicated and propagated by the host machinery. However, the possibilities of other receptors supporting viral entry (neuropilin 1, B0AT1 [neutral amino acid transporter]) should not be ruled out [15]. Because of its rapid replication, the virus damages infected organs, particularly the lungs. During the acute phase, immune cells, particularly dendritic cells and macrophages, secrete signaling molecules, which recruit other immune cells to the infected environment [11]. This process is not transient and leads to the hyperactivation of immune cells, which copiously secrete cytokines and chemokines that damage infected organs, in a response termed a “cytokine storm” [16]. Furthermore, the involvement of diverse pathways has been proposed, including cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) mediated pathways [17, 18], programmed cell death mediated by stimulation extracellular neutrophils traps (NETosis) [19], and other pathways [20]. In addition, the S protein-ACE2 interaction results in down-regulation of ACE2 on lung epithelial cells; this response is believed to be a primary cause of lung injury [21]. ACE2 simultaneously upregulates ACE1 via negative feedback mechanisms [22], thus leading to excessive angiotensin-II. Angiotensin II in turn binds angiotensin II receptor type 1 (AGTR1A) receptors and elicits excessive vascular pulmonary activity consistent with the lung pathology observed in pulmonary destruction [22, 23]. Irrespective of the signaling pathways, COVID-19 progresses through processes including lymphopenia, cytokine storms, accumulation of macrophages and neutrophils in the lungs, immune dysregulation, acute respiratory distress syndrome.

EMERGENCE OF SARS-COV-2 VARIANTS

The human immune system is tightly regulated during infection and exhibits long-lasting antigen/pathogen-specific memory over time [24–26]. To evade the host immune system, continually evolving viruses may either hijack antiviral pathways or produce diverse mutations increasing genetic diversity. These mutations can lead to significant changes in viral protein structure, including antigenic drift, folding/masking of critical antigenic residues exposed to the host, and production of new protein sequences [27, 28]. Multiple new SARS-CoV-2 variants have arisen worldwide, including the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Epsilon (B.1.427 and B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B.1.621, B.1.621.1), Zeta (P.2), Theta (P.3), and Omicron (B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5) variants (Fig 1) [29–32]. Changes in viral S protein alter the viral receptor binding pattern in the host, thus enhancing the infection potential, evading conventional detection methods, producing diverse disease outcomes, and decreasing susceptibility to the immune response raised against earlier circulating SARS-CoV-2 strains or vaccines [33]. The emergence of these variants has posed continuing challenges to host immunity induced by first-generation vaccines or prior infection.

FIGURE 1 |

Emerging SARS-CoV-2 variants. Emergence of SARS-CoV-2 variants of concern and variants of interest (https://www.who.int/activities/tracking-SARS-CoV-2-variants).

FIRST-GENERATION COVID-19 VACCINES

Despite causing unusual economic and livelihood loss globally, the COVID-19 pandemic has also driven remarkable scientific and technological progress in the past several years. A complete SARS-CoV-2 genome was quickly determined [34], and structural elucidation was performed by mapping the host receptor interactions [35]. Substantial success was attained in vaccine development against SARS-CoV-2. More than 90 vaccine candidates are being developed worldwide through various strategies, including live inactivated vaccines, viral vector-based vaccines, recombinant subunit vaccines, and nucleic acid (DNA or RNA) vaccines [36, 37]. Among them, two mRNA-based vaccines (Pfizer-BioNTech and Moderna) (Fig 2) and an adenovirus 26-based vaccine (Johnson & Johnson) were approved by the United States Food and Drug Administration for clinical use under emergency authorization. Since then, the two mRNA vaccines have received full approval. Subsequently, additional vaccines have been approved for COVID-19, including Novavax’s adjuvanted vaccine (SARS-CoV-2 S protein with Matrix-M adjuvant). Millions of doses of these vaccines have been distributed worldwide and are being used for immunizing both infected and non-infected populations [38]. These first-generation COVID-19 vaccines have been extensively reviewed elsewhere [37, 39–41].

FIGURE 2 |

Mode of action of mRNA vaccines. mRNA encapsulation by lipid nanoparticles (LNPs) aids in targeted delivery. After the mRNA enters the cell via endocytosis, the ionizable lipids in the LNPs facilitate mRNA release into the cytosol. Host ribosomes convert the mRNA into the corresponding protein, which in turn is degraded into peptides by the proteasome. The presentation of the peptides by the major histocompatibility complex to corresponding T lymphocytes stimulates both cellular and humoral immune responses.

Despite these incredible achievements, new problems have been emerged that challenge the long-term control of the pandemic; these problems include emerging viral variants (Fig 1) with greater transmissibility and immune escape ability, and waning immunity over time in vaccinated individuals [32, 42].

Most first-generation COVID-19 vaccines approved for clinical use or under clinical development target the viral S protein, its subunits (S1 and S2) or its receptor binding domain (RBD) (Fig 3A), and are aimed at eliciting a protective neutralizing antibody response. Variants with S proteins bearing structural changes—particularly those of the Delta and Omicron strains with increased transmissibility and immune escape—have emerged and become predominant [43–50]. For example, multiple S variants have been shown to have diminished sensitivity to neutralizing antibodies induced by the first-generation S-targeting vaccines [45–47]. Clinical research has also indicated that S-targeting COVID-19 vaccines show diminished efficacy against the Delta [51] and Omicron [52] strains. Thus, novel vaccine strategies targeting conserved regions of the virus to induce broader protection against COVID-19 variants are needed.

FIGURE 3 |

Structure of SARS-CoV-2 and its nucleocapsid protein. (A) SARS-CoV-2 and its four structural proteins: S, envelope, membrane, and N proteins. Physical interaction of N with the viral RNA genome. (B) Structural organization of N protein. The N-terminal domain (NTD) and C-terminal domain (CTD) are connected by an intrinsically disordered region (IDR) known as the central linker region (LKR). In addition, the NTD and CTD are flanked by N-arm and C-tail IDRs, respectively.

SARS-COV-2 NUCLEOCAPSID (N) PROTEIN BIOLOGY

N is an important structural protein of SARS-CoV-2 and is abundantly expressed in infected cells [53]. Hundreds of copies of N are present in the viral core, which encloses the genomic RNA. Like other viral structural proteins, N protein plays a crucial role in the CoV life cycle, including mRNA transcription, cytoskeletal organization, RNA replication, packaging, and immune regulation [54]. Liquid-liquid phase separation of N protein regulates viral transcription and assembly [55]. Initial studies elucidating N protein structure by nuclear magnetic resonance have indicated that its N-terminal domain (NTD) has an overall right-handed fold structure composed of a β-sheet core with an extended central loop [56]. The protein is composed of two alpha helices and five beta sheets, containing a total of 419 amino acids. It is functionally divided into two domains: the NTD and C-terminal domain (CTD) (Fig 3). The NTD, which contains an RNA-binding domain, acts as a linker between the RNA and matrix, thus enabling virion formation. N binds the positive single stranded RNA to the ribonucleoprotein core and has a “beads-on-a-string” appearance in micrographs. The CTD assists in RNA binding and contains a dimerization sequence, which facilitates RNA synthesis via interaction with the replication-transcription complexes [57, 58]. The CTD dimerization sequence also facilitates viral genome incorporation into virions. The intrinsically disordered region (IDR), between the domains, physically links the positive viral RNA genome and matrix protein [55]. Interestingly, the presence of the IDR favors N protein proteolysis, which is a key strategy for viral proliferation [59]. The proteolytic products, particularly N₁₋₂₀₉, interact with immunophilin (i.e., Cyclophilin A) in host cells and promote the SARS-CoV-2 replication cycle [59].

Because of its abundant expression after SARS-CoV-2 infection, N protein has been used as a diagnostic marker for several antigenic tests, including IgG binding assays [60]. Conventionally, the coronavirus N protein has also been shown to modulate the host intracellular machinery and play regulatory roles during the viral life cycle. Therefore, the characteristics of domains of N-protein have been targets for antiviral development via inhibiting or blocking the RNA-binding activity or oligomerization capabilities. More detailed information can be found in comprehensive reviews published elsewhere [61, 62].

HOST IMMUNITY TO SARS-COV-2 N PROTEIN

Most patients with COVID-19 mount a specific immune response against the full length and fragments of the N protein and, to a lesser extent, against a fragment containing amino acids 300–685 of S protein [63]. A total of 82% of convalescent patients have detectable N-specific IgG 12 months after SARS-CoV-2 infection, in addition to S-specific antibodies [64]. In terms of T cell responses, data from convalescent patients have indicated that the S, M, and N proteins each account for 27%, 21%, and 11% of the total CD4⁺ response, respectively [65]. Higher frequencies of multi-cytokine production (poly-functional) by M- and N-specific CD8⁺ T cells are associated with mild COVID-19 [66]. Moreover, N-specific T cell and B cell (in terms of antibody secretion) responses have been reported in different cohorts of COVID-19 infected patients [65, 67, 68]. N induces robust both CD4⁺ and CD8⁺ T cell responses. CD4⁺ and CD8⁺ T cells have been found to recognize multiple regions of N protein in all individuals who have recovered from COVID-19 [66]. Another study comparing the T cell response to SARS-CoV-1 and SARS-CoV-2 N protein has indicated that N-specific memory T cells from SARS-CoV-1 infected individuals are long-lasting and cross-reactive with SARS-CoV-2 N protein. The N-specific memory T cells remained detectable in SARS-CoV-1 infected individuals 17 years after the SARS outbreak in 2003; moreover, these T cells showed strong cross-reactivity to N protein of SARS-CoV-2 [66].

Similarly, CD8⁺ T cells specific to an immunodominant SARS-CoV-2 N epitope cross-react with selective seasonal coronaviruses [69]. Screening of SARS-CoV-2 peptide pools has revealed that the N protein induces an immunodominant response in recovered patients with HLA-B7+ COVID-19 that is also detectable in SARS-CoV-2 unexposed donors [70]. A single N-encoded epitope that is highly conserved across circulating coronaviruses drives this immunodominant response. Notably, CD8⁺ T cell responses against the N protein epitope (N_105–113) restricted by B*07:02 demonstrate strong antiviral activity and correlate with protection against severe disease [70]. Indeed, the most immunodominant CD8⁺ T cell response known to date is the N₁₀₅ peptide presented by HLA-B*07:02, which arises from a high frequency of T cells within the naive T cell repertoire that recognize N₁₀₅, rather than from human cells previously primed with coronavirus [70]. SARS-CoV-2 infection induces strong CD4⁺ and CD8⁺ T cell responses, particularly in recovered patients with severe rather than mild disease [10, 71]. Importantly, in recovered patients with mild disease, a high N-specific CD8⁺ T cell response has been observed [71]. More detailed information on cellular immunity induced by SARS-CoV-2 infection or vaccination has been reviewed elsewhere [72].

NUCLEOCAPSID-BASED SARS-COV-2 VACCINES

Beyond the S protein, the N protein is a well-recognized dominant target of antibody and T cell responses in SARS-CoV-2-infected individuals and therefore has been suggested as a potential immunogen to augment vaccine-mediated protective immunity [73]. Despite its use as a diagnostic marker, N-specific IgG in the first week of SARS-CoV-2 infection is associated with rapid symptom resolution [74]. Although current S-protein-targeting vaccines confer strong protection against ancestral SARS-CoV-2, the emerging variants have greater immune evasion of vaccine- and/or infection-elicited S-protein-specific neutralization than prior variants [75]. Therefore, vaccines expressing N protein have potential value in vaccine development. Indeed, efforts a decade ago were aimed at investigating N protein as a potential vaccine immunogen against coronaviruses. The COVID-19 pandemic has led to new light being shed on these investigations [61, 73, 76–78]. Several N-based vaccine immunogens have been developed via various technologies.

Adenovirus type-5 (Ad5) vector expressing N protein of ancestral SARS-CoV-2 has been investigated as a vaccine candidate [79, 80]. The vaccine is immunogenic and elicits both antibody and T-cell (CD4⁺ and CD8⁺) responses in mice [79]. Ad5-N alone has been found to induce very modest protection against SARS-CoV-2 in a K18-hACE2 transgenic mouse model (expressing human angiotensin-converting enzyme 2 [hACE2] receptor). However, combining Ad5-N with Ad5 expressing S (Ad5-S) has been observed to have synergistic effects and to induce stronger protection in the mouse brain (but not the lungs) than Ad5-S alone [79]. Another independent study testing Ad5-N vaccine has observed vaccine-induced protective immunity in Syrian hamsters and K18-hACE2 mice, as evidenced by decreased animal weight loss and viral loads [81].

N protein-based subunit vaccines have also been developed and tested. SARS-CoV-2 N protein either alone or in combination with well-known adjuvants (e.g., Freund’s adjuvant, alum, or QS-21) has been shown to be immunogenic in animal models [82, 83]. A synthetic peptide vaccine comprising N epitopes (HLA class I bound cytotoxic T lymphocyte peptide) along with adjuvants (Toll-like receptor [TLR] 4 agonist (MPLA) and TLR9 agonist [CpG oligonucleotide]) have shown moderate protection against SRAS-CoV-2 in Rhesus macaques [84].

Our group has generated a nucleoside-modified, lipid-nanoparticle (LNP)-formulated mRNA vaccine that encodes the full-length N protein of SARS-CoV-2 (Wuhan-Hu-1 strain) (mRNA-N) [60]. Immunogenicity analyses in mice have indicated that mRNA-N is highly immunogenic and induces strong N-specific binding antibody and T cell (both CD4⁺ and CD8⁺) responses. As expected, no neutralizing antibody response is elicited by mRNA-N [60]. Similarly to N-expressing vaccines based on other platforms [79, 80], the mRNA-N vaccine has been found to induce modest protection against mouse-adapted SARS-CoV-2 and the Delta strain in mice and hamsters [60].

MULTIVALENT SARS-COV-2 VACCINES EXPRESSING MULTIPLE VIRAL PROTEINS

To elicit both neutralizing antibodies and broadly protective T cell responses, several multivalent SARS-CoV-2 vaccines expressing both S and N proteins have been developed, mainly through use of viral vectors. A synthetic modified vaccinia Ankara (MVA) vaccine expressing SARS-CoV-2 S and N protein (COH04S1) has been shown to be immunogenic [85] and to confer protection against a challenge with ancestral SARS-CoV-2 in non-human primates (NHPs) [86]. The efficacy of this vaccine against major SARS-CoV-2 variants remains to be determined. The vaccine has been evaluated in a phase I clinical trial, which has indicated that the vaccine is well tolerated and elicits S- and N-specific immune responses in healthy participants [87]. Similarly, another independent study has investigated the effect of a recombinant MVA vaccine expressing S and N proteins (MVA/SdFCS-N) in NHPs against the SARS-CoV-2 Delta variant [88]. Among the different routes of administration, MVA/SdFCS-N administration via an intramuscular route has been found to better protection against the Delta variant than other routes [88].

Another study has focused on the development of trivalent COVID-19 vaccines by using adenoviral vectors of human and chimpanzee origin expressing the S1, N, and RNA-dependent RNA polymerase of SARS-CoV-2 [89] and also has compared intramuscular and intranasal immunization strategies. Of note, a single intranasal mucosal immunization led to strong, systemic neutralizing antibodies. In addition, intranasal vaccination increased tissue resident memory CD8⁺ T cells in the respiratory mucosa as well as locally trained macrophages. The intranasal strategy conferred protection against the parent strain of SARS-CoV-2 as well as other variants of concern (B.1.1.7 and B.1.351) [89].

Using the human Ad5 vector, ImmunityBio has developed an approach targeting both SARS-CoV-2 S and N proteins. The S and Enhanced T-cell Stimulation Domain (N-ETSD) of the N protein are used together in the vaccine, and both have been found to be immunogenic in mice. Subcutaneous priming and intranasal or subcutaneous boosting with this dual antigen vaccine induces greater Th1-biased T-cell and humoral responses than Ad5-S alone [90]. In a phase I trial, a single dose (1 × 10¹¹ viral particle) vaccination in healthy adults has been found to elicit a strong T cell response [91]. This platform supports the feasibility of needleless immunizations in general. However, viral challenge studies are needed to evaluate the vaccine efficacy in animal models.

Vesicular stomatitis virus is a commonly used vector for mucosal delivery. Intranasal administration of a vesicular stomatitis virus-based vaccine expressing S and N proteins has been found to elicit a mucosal immune response and protective effects in a hamster viral challenge model, whereas the same vaccine administered intramuscularly is only marginally protective [92].

In addition to viral vectors, multivalent vaccines based on DNA or proteins have been reported. Mice immunized with a DNA vaccine encoding S/RBD and N protein have been found to develop broad neutralizing antibodies and N-specific T cell responses [93]. The DNA vaccine has been found to protect mice against lethal SARS-CoV-2 infection [93]. Similarly, a phase I clinical trial of a DNA vaccine (GX-19N™) encoding the RBD and N protein has demonstrated its safety and immunogenicity [94], although the efficacy of this vaccine in animal models is unclear. Hong and colleagues have immunized macaques with multivalent protein subunit vaccine comprising the RBD fused with tetanus toxoid epitope P2 (RBD-P2) and N protein, and shown that addition of N protein induces slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 alone in NHPs [95].

Although the above multivalent vaccine approaches have been found to elicit various levels of protection against SARS-CoV-2 or its variants in different animal models, a direct comparison of their efficacy in protecting against SARS-CoV-2 VOCs, with respect to the efficacy of current clinically approved first-generation COVID-19 vaccines, particularly the mRNA-S vaccines, is critically lacking. Their efficacy against the currently predominant Omicron variants also remains to be determined through comparison with the clinically approved S-protein-targeting vaccines.

As discussed earlier, the mRNA vaccine expressing the full-length N protein (mRNA-N) generated by our group confers modest protection against a mouse adaptive SARS-CoV-2 and Delta strain. To enable comparison with first-generation S-protein-targeting vaccines, we also generated an mRNA-LNP vaccinee expressing the prefusion-stabilized SARS-CoV-2 S protein (mRNA-S), whose immunogenicity and protective efficacy against multiple SARS-CoV-2 VOCs was evaluated either alone or in combination with mRNA-N (mRNA-S+N) in rodent models [60]. Our data showed that mRNA S+N vaccination induced markedly stronger protection than mRNA-S alone against multiple SARS-CoV-2 strains, including a mouse-adapted strain, Delta, and Omicron, in both the lower and upper respiratory tracts. The difference between mRNA-S alone and mRNA-S+N was most profound in the protection against Omicron: whereas mRNA-S alone had a substantially diminished efficacy against Omicron because of strong immune escape, mRNA-S+N vaccination conferred substantial protection against Omicron (four of five animals had no detectable viral RNA and titers in the lungs) [60], thus indicating induction of cross-protective immunity against VOCs by mRNA-S+N. In vivo CD8⁺ T cell depletion supports that CD8⁺ T cells play a critical role in protection against VOCs after mRNA-S+N vaccine immunization [60]. Collectively, our findings suggest that the bivalent mRNA-S+N is a potential pan-COVID-19 vaccine for emerging SARS-CoV-2 variants.

SUMMARY AND FUTURE DIRECTIONS

As new variants of SARS-CoV-2 continue to arise, pan-COVID-19 vaccines that induce broad protection against emerging strains must urgently be developed. Current strategies to develop vaccines against COVID-19 VOCs, include utilization of VOC-specific S immunogens. Although a VOC-targeted S protein booster has been clinically approved in the two mRNA vaccinees, this strategy is likely to be challenging, because as S proteins continually mutate, the design and selection of VOC-specific sequences will be become less optimal. Given the more conserved sequence of N than S protein across different variants, as well as the cross-reactive, long-lasting N-protein-specific T cell immunity [96, 97], our study and those from other groups have clearly demonstrated the benefit and utility of including N immunogen in the next-generation COVID-19 vaccines for VOCs. Our study has indicated that, despite the use of ancestral sequences, the inclusion of mRNA-N along with mRNA-S for vaccination elicits a robust protection against both the Delta and Omicron strains, which is not achieved by mRNA-S alone. Given the demonstrated safety profile of the mRNA-LNP platform in large human populations, the bivalent mRNA-S+N vaccine should be advanced to clinical testing.

Several important questions remain to be explored. First, the durability of vaccine-elicited protective immunity is critical [98, 99]. Whether the dual mRNA-S+N vaccine induces more durable protective immunity than mRNA-S alone is unclear and is currently under investigation. Second, because a large human population has been vaccinated with first-generation vaccines or naturally infected with the virus, further testing the inclusion of N immunogen as a booster strategy will be likely to reveal additional insights into the utility of this bivalent vaccine approach against SARS-CoV-2 VOCs. Third, further validating the safety and efficacy of the vaccine approach in larger animal models will be helpful before clinical testing. Finally, because COVID-19 VOCs can infect individuals through vaccine breakthrough and antibody escape, screening for broadly neutralizing or pan-neutralizing antibodies against COVID-19 VOCs is also important for the development of a pan-COVID-19 vaccine, as well as the prevention and treatment of VOC infections in the future.

CONFLICTS OF INTEREST

No conflicts of interest are declared by the authors.

REFERENCES

1. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al.. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020. Vol. 382:727–733

2. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al.. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020. Vol. 579:270–273

3. World Health Organization. 2020. Vol. Vol. 2023. https://www.who.int/director-general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march-2020

4. World Health Organization. 2022. https://www.who.int/publications/m/item/covid-19-weekly-epidemiological-update---21-december-2022

5. Salzberger B, Buder F, Lampl B, Ehrenstein B, Hitzenbichler F, Holzmann T, et al.. Epidemiology of SARS-CoV-2. Infection. 2021. Vol. 49(2):233–239

6. Pijls BG, Jolani S, Atherley A, Derckx RT, Dijkstra JIR, Franssen GHL, et al.. Demographic risk factors for COVID-19 infection, severity, ICU admission and death: a meta-analysis of 59 studies. BMJ Open. 2021. Vol. 11(1):e044640

7. Osman M, Klopfenstein T, Belfeki N, Gendrin V, Zayet S. A comparative systematic review of COVID-19 and influenza. Viruses. 2021. Vol. 13:452

8. Wiersinga WJ, Rhodes A, Cheng AC, Peacock SJ, Prescott HC. Pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (COVID-19): a review. JAMA. 2020. Vol. 324:782–793

9. de Almeida-Pititto B, Dualib PM, Zajdenverg L, Dantas JR, de Souza FD, Rodacki M, et al.. Severity and mortality of COVID 19 in patients with diabetes, hypertension and cardiovascular disease: a meta-analysis. Diabetol Metab Syndr. 2020. Vol. 12:75

10. Saksena N, Bonam SR, Miranda-saksena M, Cardoso TH. Incursions by severe acute respiratory syndrome coronavirus-2 on the host anti-viral immunity during mild, moderate, and severe coronavirus disease 2019 disease. Explor Immunol. 2022. Vol. 2:794–811

11. Bonam SR, Kotla NG, Bohara RA, Rochev Y, Webster TJ, Bayry J. Potential immuno-nanomedicine strategies to fight COVID-19 like pulmonary infections. Nano Today. 2021. Vol. 36:101051

12. Saksena N, Bonam SR, Miranda-Saksena M. Epigenetic lens to visualize the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in COVID-19 pandemic. Front Genet. 2021. Vol. 12:581726

13. Saksena N, Bonam SR, Miranda-Saksena M. Immunopathogenesis of severe acute respiratory syndrome coronavirus-2: evolving knowledge and its current status. Explor Immunol. 2021. Vol. 1:61–79

14. Xu H, Zhong L, Deng J, Peng J, Dan H, Zeng X, et al.. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci. 2020. Vol. 12:8

15. Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol. 2022. Vol. 23:3–20

16. Fajgenbaum DC, June CH. Cytokine storm. New Engl J Med. 2020. Vol. 383:2255–2273

17. Hong W, Chen Y, You K, Tan S, Wu F, Tao J, et al.. Celebrex adjuvant therapy on coronavirus disease 2019: an experimental study. Front Pharmacol. 2020. Vol. 11:561674

18. Tomera K, Kittah J. Rapid clinical recovery from severe COVID-19 with high dose famotidine and high dose celecoxib adjuvant therapy. Preprints. 2020. 2020080519. [Cross Ref]

19. Arcanjo A, Logullo J, Menezes CCB, de Souza Carvalho Giangiarulo TC, Dos Reis MC, de Castro GMM, et al.. The emerging role of neutrophil extracellular traps in severe acute respiratory syndrome coronavirus 2 (COVID-19). Sci Rep. 2020. Vol. 10:19630

20. Dotan A, David P, Arnheim D, Shoenfeld Y. The autonomic aspects of the post-COVID19 syndrome. Autoimmun Rev. 2022. Vol. 21:103071

21. Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, et al.. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013. Vol. 503(7477):535–538

22. Cao YN, Li L, Feng Z, Wan S, Huang P, Sun X, et al.. Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discov. 2020. Vol. 6:11

23. Kakodkar P, Kaka N, Baig MN. A comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease 2019 (COVID-19). Cureus. 2020. Vol. 12:e7560

24. Rouse BT, Sehrawat S. Immunity and immunopathology to viruses: what decides the outcome? Nat Rev Immunol. 2010. Vol. 10:514–526

25. Bonam SR, Partidos CD, Halmuthur SKM, Muller S. An overview of novel adjuvants designed for improving vaccine efficacy. Trends Pharmacol Sci. 2017. Vol. 38:771–793

26. Pichlmair A, Reis e Sousa C. Innate recognition of viruses. Immunity. 2007. Vol. 27:370–383

27. Yewdell JW. Antigenic drift: understanding COVID-19. Immunity. 2021. Vol. 54:2681–2687

28. Cao Y, Yisimayi A, Jian F, Song W, Xiao T, Wang L, et al.. BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature. 2022. Vol. 608(7923):593–602

29. Dietz L, Horve PF, Coil DA, Fretz M, Eisen JA, Van Den Wymelenberg K. 2019 Novel coronavirus (COVID-19) pandemic: built environment considerations to reduce transmission. mSystems. 2020. Vol. 5(2):e00245–00220

30. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, et al.. Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell. 2020. Vol. 182:812–827.e819

31. Centers for Disease Control and Prevention. 2022. Vol. Vol. 2022. https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-classifications.html

32. Harvey WT, Carabelli AM, Jackson B, Gupta RK, Thomson EC, Harrison EM, et al.. SARS-CoV-2 variants, spike mutations and immune escape. Nat Rev Microbiol. 2021. Vol. 19:409–424

33. Frank F, Keen MM, Rao A, Bassit L, Liu X, Bowers HB, et al.. Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests. Cell. 2022. Vol. 185(19):3603–3616.e13

34. Wang H, Li X, Li T, Zhang S, Wang L, Wu X, et al.. The genetic sequence, origin, and diagnosis of SARS-CoV-2. Eur J Clin Microbiol Infect Dis. 2020. Vol. 39(9):1629–1635

35. Yang J, Petitjean SJL, Koehler M, Zhang Q, Dumitru AC, Chen W, et al.. Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor. Nat Commun. 2020. Vol. 11:4541

36. Andreadakis Z, Le TT, Kumar A, Román RG, Tollefsen S, Saville M, et al.. The COVID-19 vaccine development landscape. Nat Rev Drug Discov. 2020. Vol. 19:305–306

37. Krammer F. SARS-CoV-2 vaccines in development. Nature. 2020. Vol. 586:516–527

38. Chaudhary N, Weissman D, Whitehead KA. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Dis. 2021. Vol. 20:817–838

39. Kyriakidis NC, Lopez-Cortes A, Gonzalez EV, Grimaldos AB, Prado EO. SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates. NPJ Vaccines. 2021. Vol. 6:28

40. Li Y, Tenchov R, Smoot J, Liu C, Watkins S, Zhou Q. A comprehensive review of the global efforts on COVID-19 vaccine development. ACS Cent Sci. 2021. Vol. 7:512–533

41. Barouch DH. Covid-19 vaccines - immunity, variants, boosters. N Engl J Med. 2022. Vol. 387:1011–1020

42. Shah M, Woo HG. The paradigm of immune escape by SARS-CoV-2 variants and strategies for repositioning subverted mAbs against escaped VOCs. Mol Ther. 2022. Vol. 30:3101–3105

43. Madhi SA, Baillie V, Cutland CL, Voysey M, Koen AL, Fairlie L, et al.. Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against the B.1.351 Variant. N Engl J Med. 2021. Vol. 384:1885–1898

44. Moore JP, Offit PA. SARS-CoV-2 vaccines and the growing threat of viral variants. JAMA. 2021. Vol. 325:821–822

45. Dejnirattisai W, Zhou D, Supasa P, Liu C, Mentzer AJ, Ginn HM, et al.. Antibody evasion by the P.1 strain of SARS-CoV-2. Cell. 2021. Vol. 184:2939–2954.e2939

46. Zhou D, Dejnirattisai W, Supasa P, Liu C, Mentzer AJ, Ginn HM, et al.. Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera. Cell. 2021. Vol. 184:2348–2361.e2346

47. Garcia-Beltran WF, Lam EC, St Denis K, Nitido AD, Garcia ZH, Hauser BM, et al.. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell. 2021. Vol. 184(9):2372–2383.e2379

48. Cao Y, Wang J, Jian F, Xiao T, Song W, Yisimayi A, et al.. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature. 2022. Vol. 602(7898):657–663

49. Planas D, Saunders N, Maes P, Guivel-Benhassine F, Planchais C, Buchrieser J, et al.. Considerable escape of SARS-CoV-2 Omicron to antibody neutralization. Nature. 2022. Vol. 602(7898):671–675

50. Liu L, Iketani S, Guo Y, Chan JF, Wang M, Liu L, et al.. Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2. Nature. 2022. Vol. 602(7898):676–681

51. Tang P, Hasan MR, Chemaitelly H, Yassine HM, Benslimane FM, Al Khatib HA, et al.. BNT162b2 and mRNA-1273 COVID-19 vaccine effectiveness against the SARS-CoV-2 Delta variant in Qatar. Nat Med. 2021. Vol. 27(12):2136–2143

52. Andrews N, Stowe J, Kirsebom F, Toffa S, Rickeard T, Gallagher E, et al.. Covid-19 vaccine effectiveness against the omicron (B.1.1.529) variant. N Engl J Med. 2022. Vol. 386(16):1532–1546

53. He Y, Zhou Y, Wu H, Kou Z, Liu S, Jiang S. Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus. J Clin Microbiol. 2004. Vol. 42(11):5309–5314

54. Cong Y, Ulasli M, Schepers H, Mauthe M, V’kovski P, Kriegenburg F, et al.. Nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle. J Virol. 2020. Vol. 94:e01925–e01919

55. Savastano A, Ibáñez de Opakua A, Rankovic M, Zweckstetter M. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates. Nat Commun. 2020. Vol. 11:6041

56. Dinesh DC, Chalupska D, Silhan J, Koutna E, Nencka R, Veverka V, et al.. Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein. PLoS Path. 2020. Vol. 16:e1009100

57. Lu S, Ye Q, Singh D, Cao Y, Diedrich JK, Yates JR, et al.. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein. Nat Commun. 2021. Vol. 12:502

58. Cubuk J, Alston JJ, Incicco JJ, Singh S, Stuchell-Brereton MD, Ward MD, et al.. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat Commun. 2021. Vol. 12(1):1936

59. Lutomski CA, El-Baba TJ, Bolla JR, Robinson CV. Multiple roles of SARS-CoV-2 N protein facilitated by proteoform-specific interactions with RNA, host proteins, and convalescent antibodies. JACS Au. 2021. Vol. 1(8):1147–1157

60. Hajnik RL, Plante JA, Liang Y, Alameh MG, Tang J, Bonam SR, et al.. Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Sci Transl Med. 2022. Vol. 14(662):eabq1945

61. Chang Ck, Hou MH, Chang CF, Hsiao C-D, Huang T-h. The SARS coronavirus nucleocapsid protein – Forms and functions. Antiviral Res. 2014. Vol. 103:39–50

62. Matsuo T. Viewing SARS-CoV-2 nucleocapsid protein in terms of molecular flexibility. Biology. 2021. Vol. 10(6):454

63. Smits VAJ, Hernández-Carralero E, Paz-Cabrera MC, Cabrera E, Hernández-Reyes Y, Hernández-Fernaud JR, et al.. The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients. Biochem Biophys Res Commun. 2021. Vol. 543:45–49

64. Guo L, Wang G, Wang Y, Zhang Q, Ren L, Gu X, et al.. SARS-CoV-2-specific antibody and T-cell responses 1 year after infection in people recovered from COVID-19: a longitudinal cohort study. Lancet Microbe. 2022. Vol. 3(5):e348–e356

65. Grifoni A, Weiskopf D, Ramirez SI, Mateus J, Dan JM, Moderbacher CR, et al.. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell. 2020. Vol. 181(7):1489–1501.e1415

66. Le Bert N, Tan AT, Kunasegaran K, Tham CYL, Hafezi M, Chia A, et al.. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020. Vol. 584(7821):457–462

67. Ni L, Ye F, Cheng ML, Feng Y, Deng YQ, Zhao H, et al.. Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals. Immunity. 2020. Vol. 52(6):971–977.e973

68. Woo PCY, Lau SK, Wong BH, Tsoi HW, Fung AM, Chan KH, et al.. Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol. 2004. Vol. 42(5):2306–2309

69. Lineburg KE, Grant EJ, Swaminathan S, Chatzileontiadou DSM, Szeto C, Sloane H, et al.. CD8(+) T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses. Immunity. 2021. Vol. 54(5):1055–1065.e1055

70. Nguyen THO, Rowntree LC, Petersen J, Chua BY, Hensen L, Kedzierski L, et al.. CD8(+) T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity. Immunity. 2021. Vol. 54(5):1066–1082.e1065

71. Peng Y, Mentzer AJ, Liu G, Yao X, Yin Z, Dong D, et al.. Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19. Nat Immunol. 2020. Vol. 21(11):1336–1345

72. Moss P. The T cell immune response against SARS-CoV-2. Nat Immunol. 2022. Vol. 23:186–193

73. Dutta NK, Mazumdar K, Gordy JT. The nucleocapsid protein of SARS-CoV-2: a target for vaccine development. J Virol. 2020. Vol. 94(13):e00647–e00720

74. Jia X, Cao S, Lee AS, Manohar M, Sindher SB, Ahuja N, et al.. Anti-nucleocapsid antibody levels and pulmonary comorbid conditions are linked to post–COVID-19 syndrome. JCI Insight. 2022. Vol. 7(13):e156713

75. Kurhade C, Zou J, Xia H, Liu M, Chang HC, Ren P, et al.. Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1, and XBB.1 by parental mRNA vaccine or a BA.5-bivalent booster. Nat Med. 2023d. Vol. 29(2):344–347

76. Sheikh A, Al-Taher A, Al-Nazawi M, Al-Mubarak AI, Kandeel M. Analysis of preferred codon usage in the coronavirus N genes and their implications for genome evolution and vaccine design. J Virol Methods. 2020. Vol. 277:113806

77. Dutta NK, Mazumdar K, Lee BH, Baek MW, Kim DJ, Na YR, et al.. Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine. Immunol Lett. 2008. Vol. 118(1):65–71

78. Wu W, Cheng Y, Zhou H, Sun C, Zhang S. 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. Virol J. 2023. Vol. 20(1):6

79. Dangi T, Class J, Palacio N, Richner JM, Penaloza MacMaster P. Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2. Cell Rep. 2021. Vol. 36(10):109664

80. Dangi T, Sanchez S, Class J, Richner M, Visvabharathy L, Chung YR, et al.. Improved control of SARS-CoV-2 by treatment with a nucleocapsid-specific monoclonal antibody. J Clin Invest. 2022. Vol. 132(23):e162282

81. Matchett WE, Joag V, Stolley JM, Shepherd FK, Quarnstrom CF, Mickelson CK, et al.. Cutting edge: nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity. J Immunol. 2021. Vol. 207(2):376–379

82. Silva EKVB, Bomfim CG, Barbosa AP, Noda P, Noronha IL, Fernandes BHV, et al.. Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats. PLoS One. 2022. Vol. 17(5):e0268434

83. Feng W, Xiang Y, Wu L, Chen Z, Li Q, Chen J, et al.. Nucleocapsid protein of SARS-CoV-2 is a potential target for developing new generation of vaccine. J Clin Lab Anal. 2022. Vol. 36(6):e24479

84. Harris PE, Brasel T, Massey C, Herst CV, Burkholz S, Lloyd P, et al.. A synthetic peptide CTL vaccine targeting nucleocapsid confers protection from SARS-CoV-2 challenge in rhesus macaques. Vaccines (Basel). 2021. Vol. 9(5):520

85. Chiuppesi F, Salazar MD, Contreras H, Nguyen VH, Martinez J, Park Y, et al.. Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform. Nat Commun. 2020. Vol. 11(1):6121

86. Chiuppesi F, Nguyen VH, Park Y, Contreras H, Karpinski V, Faircloth K, et al.. Synthetic multiantigen MVA vaccine COH04S1 protects against SARS-CoV-2 in Syrian hamsters and non-human primates. NPJ Vaccines. 2022. Vol. 7(1):7

87. Chiuppesi F, Zaia JA, Frankel PH, Stan R, Drake J, Williams B, et al.. Safety and immunogenicity of a synthetic multiantigen modified vaccinia virus Ankara-based COVID-19 vaccine (COH04S1): an open-label and randomised, phase 1 trial. Lancet Microbe. 2022. Vol. 3(4):e252–e264

88. Routhu NK, Gangadhara S, Lai L, Davis-Gardner ME, Floyd K, Shiferaw A, et al.. A modified vaccinia Ankara vaccine expressing spike and nucleocapsid protects rhesus macaques against SARS-CoV-2 Delta infection. Sci Immunol. 2022. Vol. 7(72):eabo0226

89. Afkhami S, D’Agostino MR, Zhang A, Stacey HD, Marzok A, Kang A, et al.. Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2. Cell. 2022. Vol. 185(5):896–915.e19

90. Rice A, Verma M, Shin A, Zakin L, Sieling P, Tanaka S, et al.. Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice. Sci Rep. 2021. Vol. 11(1):14917

91. Sieling P, King T, Wong R, Nguyen A, Wnuk K, Gabitzsch E, et al.. Prime hAd5 spike + nucleocapsid vaccination induces ten-fold increases in mean T-cell responses in phase 1 subjects that are sustained against spike variants. medRxiv. 2021. 2021.2004.2005.21254940

92. O’Donnell KL, Gourdine T, Fletcher P, Clancy CS, Marzi A. Protection from COVID-19 with a VSV-based vaccine expressing the spike and nucleocapsid proteins. Front Immunol. 2022. Vol. 13:1025500

93. Appelberg S, Ahlén G, Yan J, Nikouyan N, Weber S, Larsson O, et al.. A universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells. EMBO Mol Med. 2022. Vol. 14(10):e15821

94. Ahn JY, Lee J, Suh YS, Song YG, Choi YJ, Lee KH, et al.. Safety and immunogenicity of two recombinant DNA COVID-19 vaccines containing the coding regions of the spike or spike and nucleocapsid proteins: an interim analysis of two open-label, non-randomised, phase 1 trials in healthy adults. Lancet Microbe. 2022. Vol. 3(3):e173–e183

95. Hong SH, Oh H, Park YW, Kwak HW, Oh EY, Park HJ, et al.. Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates. Sci Adv. 2021. Vol. 7(22):eabg7156

96. Zhao J, Zhao J, Mangalam AK, Channappanavar R, Fett C, Meyerholz DK, et al.. Airway memory CD4(+) T cells mediate protective immunity against emerging respiratory coronaviruses. Immunity. 2016. Vol. 44(6):1379–1391

97. McKinstry KK, Strutt TM, Kuang Y, Brown DM, Sell S, Dutton RW, et al.. Memory CD4+ T cells protect against influenza through multiple synergizing mechanisms. J Clin Invest. 2012. Vol. 122(8):2847–2856

98. Qu P, Faraone JN, Evans JP, Zheng YM, Yu L, Ma Q, et al.. Durability of booster mRNA vaccine against SARS-CoV-2 BA.2.12.1, BA.4, and BA.5 subvariants. N Engl J Med. 2022. Vol. 387(14):1329–1331

99. Townsend JP, Hassler HB, Sah P, Galvani AP, Dornburg A. The durability of natural infection and vaccine-induced immunity against future infection by SARS-CoV-2. Proc Natl Acad Sci U S A. 2022. Vol. 119(31):e2204336119