key: cord-0942135-umegu13v
authors: Arashkia, Arash; Jalilvand, Somayeh; Mohajel, Nasir; Afchangi, Atefeh; Azadmanesh, Kayhan; Salehi‐Vaziri, Mostafa; Fazlalipour, Mehdi; Pouriayevali, Mohammad Hassan; Jalali, Tahmineh; Mousavi Nasab, Seyed Dawood; Roohvand, Farzin; Shoja, Zabihollah
title: Severe acute respiratory syndrome‐coronavirus‐2 spike (S) protein based vaccine candidates: State of the art and future prospects
date: 2020-10-15
journal: Rev Med Virol
DOI: 10.1002/rmv.2183
sha: 7b880c7467b893c71154bc3933097fddf5b31b8c
doc_id: 942135
cord_uid: umegu13v

Coronavirus disease 2019 (Covid‐19) is caused by severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) which is responsible for a global pandemic that started in late 2019 in Wuhan, China. To prevent the worldwide spread of this highly pathogenic virus, development of an effective and safe vaccine is urgently needed. The SARS‐CoV‐2 and SARS‐CoV share a high degree of genetic and pathologic identity and share safety and immune‐enhancement concerns regarding vaccine development. Prior animal studies with first generation (whole virus‐based) preparations of SARS‐CoV vaccines (inactivated and attenuated vaccine modalities) indicated the possibility of increased infectivity or eosinophilic infiltration by immunization. Therefore, development of second and third generation safer vaccines (by using modern vaccine platforms) is actively sought for this viral infection. The spike (S) protein of SARS‐CoVs is the main determinant of cell entry and tropism and is responsible for facilitating zoonosis into humans and sustained person‐to‐person transmission. Furthermore, ‘S’ protein contains multiple neutralizing epitopes that play an essential role in the induction of neutralizing antibodies (nAbs) and protective immunity. Moreover, T‐cell responses against the SARS‐CoV‐2 ‘S’ protein have also been characterized that correlate to the IgG and IgA antibody titres in Covid‐19 patients. Thus, S protein is an obvious candidate antigen for inclusion into vaccine platforms against SARS‐CoV‐2 viral infection. This manuscript reviews different characteristics of S protein, its potency and ‘state of the art’ of the vaccine development strategies and platforms using this antigen, for construction of a safe and effective SARS‐CoV‐2 vaccine.

was named SARS-CoV-2 and the disease caused by SARS-

CoV-2 is believed to have originated from bats, but pangolins are proposed as possible intermediate hosts. 12 Although the mortality rates in SARS-CoV-2 infection are not as high compared to that of the SARS-CoV and MERS-CoV, SARS-CoV-2 is more transmissible and so has claimed considerably more lives. 1 Additionally, the newly reported D614G amino acid change in spike (S) protein seems to have augmented infectivity of the SARS-CoV-2. 13 Development of a vaccine against this viral infection is the priority of WHO and other global healthcare organizations. However, several drugs are being evaluated for efficacy in treating SARS-CoV-2, among which remdesivir and dexamethasone have shown improved outcomes in very ill patients. 14, 15 The results of recent randomized clinical trials (RCTs) demonstrated that remdesivir (that received FDA authorization of emergency use in severe Covid-19 patients) [16] [17] [18] and dexamethasone 19 can decrease the recovery time for Covid-19 hospitalized-patients under supplemental, oxygen therapy. There are also ongoing RCTs evaluating the safety and efficacy of the immunomodulator interferon beta-1a alone (NCT04385095) or in combination with remdesivir (NCT04492475).

The SARS-CoV-2 genome encodes several non-structural (NSP1-NSP10 and NSP12-16) and accessory proteins as well as four structural proteins, including spike (S), envelope (E), membrane (M) and nucleocapsid (N; Figure 1a ). Among structural proteins, S, is responsible for binding to cellular-angiotensin-converting enzyme 2 (ACE2; which acts as the cellular receptor) and thus is an obvious candidate antigen for vaccine development based on induction of neutralizing antibodies (nAbs) against the virus. 11, 20 Phylogenetic analysis of full-length genomes indicated that SARS-CoV-2 is more closely related to bat-SL-CoV ZC45 and bat-SL-CoV ZXC21 (but more distantly related to SARS-CoV) 11 and maximum homology (96.2% nucleotide sequence identity) with CoV RaTG13 isolated from Rhinolophus affinis bats. 21 Interestingly however, in phylogenetic analyses based on receptor-binding domain (RBD) of the S or the S gene regions, SARS-CoV-2 was more closely related to SARS-CoV, indicating the high sequence similarity of S gene between two viruses. 11 Accordingly, the origination of SARS-CoV-2 is commonly believed to be through the recombination of bat SARS-CoVs with most frequent recombination breakpoints located within the 'S' gene. 22 To date, there are two propositions to explain the origin of SARS-CoV-2.

The first scenario is based on high genomic sequence similarity (96%) between SARS-CoV-2 and the CoV isolated from a bat in 2013 (bat CoV RaTG13) and suggests a possible homologous recombination between the bat CoV and another CoV of unknown origin. 23 The second scenario is based on natural selection in humans following zoonotic transmission. 21, 24 Indeed, S protein plays an essential role in viral attachment, fusion and entry into the host cells and might be the key protein for crossing the species barrier for adaptive evolution and animal-to-human transmission of SARS-CoVs. 25, 26 It is shown that nAbs targeting S protein block virus interaction with ACE2, while T-cell responses against the SARS-CoV-2 S protein correlate with IgG and IgA antibody titres in Covid-19 patients. [27] [28] [29] [30] Therefore, the S protein has attracted particular attention as the most likely target antigen for long-term immune response and vaccine design to SARS-CoV-2.

The present manuscript, reviews different characteristics of S protein, its potency and 'state of the art' of the vaccine development strategies and platforms using this antigen, for construction of a safe and effective SARS-CoV-2 vaccine.

The S gene encodes a 1273 amino acid protein which is heavily glycosylated during its synthesis and assembles into trimers on the virion surface, resulting to the crown-like appearance or corona. 31 Schematic diagram of the S protein and its various domains is presented in Figure 1b . Two functional subunits S1 and S2 that arise from proteolytic processing are responsible for binding to the host cell receptor and fusion, respectively. Although the S1 subunits of SARS-CoV and SARS-CoV-2 can bind ACE2 to infect humans, the affinity of the RBD in the S1 subunit to ACE2 in SARS-CoV-2 is 10 to 20 fold stronger than that of the SARS-CoV, which may contribute to the higher spread rate of SARS-CoV-2 from human to human. 32 Unlike SARS-CoV, the S protein of SARS-CoV-2 contains a polybasic four residues at the boundary between the S1 and S2 subunits (a furin cleavage site) that might contribute to the tropism and transmissibility of SARS-CoV-2. 21 The cryo-electron microscopy (Cryo-EM) data of SARS-CoV and MERS-CoV S proteins indicated that the binding of S1 subunit to the host cell receptor forms a metastable pre-fusion conformation ('up'/ 'opened' conformation and /or 'down'/'closed' conformation) that switches a stable post-fusion conformation in the S2 subunit to facilitate the fusion steps. Such up and down conformations might be responsible for receptor-accessible and receptor-inaccessible states, respectively. Accordingly, recent studies on SARS-CoV-2 ( Figure 2) indicated the presence of trimers with only a single RBD up protomer. 25, 32 This finding suggests that unstable, distinct conformational states might lead to the initiation of fusogenic conformational change similar to the highly pathogenic CoVs (SARS-CoV and MERS-CoV). 33, 34 This is in contrast to the common cold-related CoVs that have RBD down conformation in the S trimers. [35] [36] [37] [38] It should be noted however that in case of HCoV-NL63 and HCoV-229E with closed S trimers, RBDs hidden at the interface between protomers might need to be exposed. 39, 40 Overall, these findings emphasize that S protein trimers in highly pathogenic CoVs seem to exist in partially opened (up) state, whereas they remain largely closed (down) in CoVs associated with the common cold.

The SARS-CoV-2 S protein contains 22 N-linked glycosylation sequons per protomer that contain oligomannose and complex glycans ( Figure 1b) . Glycosylation is critical to folding of S glycoprotein and immune evasion by shielding specific epitopes from antibody neutralization. Of note, several proximal glycosylation sites (N165, N234, N343) are able to mask RBD on S trimer, especially in RBD closed or down conformation. 25, 41 

Despite uncertainty about immunological correlates of protection for Covid-19, correlation of virus-specific nAbs titres and the numbers of virus-specific T cells to SARS-CoV-2 (specially against S protein) with effective clearance of virus is reported in several studies (outlined in the following).

The coding sequence for SARS-CoV-2 proteins. The orf1ab encodes the pp1ab protein that contains 15 nsps (nsp1-nsp10 and nsp12-nsp16). The orf1a encodes the pp1a protein that contains ten nsps (nsp1-nsp10). SARS-CoV-2 encodes four structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N) and eight accessory proteins 3a, 3b, p6, 7a, 7b, 8b, 9b, and ORF14. 20 

It is well known that the humoral immune response is the critical primary effector of protective immunity for natural viral infection and vaccines. In case of Covid-19, seroconversion in most of the infected people occur between 7 and 14 days after the onset of symptoms, starting with the detection of IgM and IgA antibodies (that can be detected early during the first week or 3 weeks of symptom onset) followed by IgG detection by around 14 days after the initiation of symptoms 28, 42, 43 (Figure 3 ). Rise in the Ab levels is also accompanied by the increase in activated CD4þ/CD8þ T-cells and plasma cells in peripheral blood mononuclear cells (PBMCs) 44, 45 while IgG memory cells specific to the RBD have also been detected in the blood of Covid-19 patients. 46 Similarly, the prevention of reinfection in SARS-CoV-2 infected rhesus macaques correlated with the rise of antibodies in recovered animals. 47 In parallel, several studies in infected patients have shown the presence of serum IgA against SARS-CoV-2 with neutralizing potential 42, 48 as shown previously in preclinical animals studies (in bronchoalveolar lavages) with SARS-CoV vaccine candidates. 49, 50 In general antibodies against both the N and S proteins are commonly detectable, among which those raised against RBD of S protein can be potently neutralizing and could be detected in most tested Covid-19 patients. 28, 46, 51 Of note, neither plasma of convalescent Covid-19 patients nor SARS-CoV-2 RBD-specific neutralizing monoclonal antibodies (mAbs) showed any cross-reactivity with that of the SARS-CoV or MERS-CoV. However, that of the SARS-CoV showed cross-reactive neutralization with SARS-CoV-2 26,52-54 indicating the possibility of using SARS-CoV S (RBD) as the antigen to induce nAbs against SARS-CoV-2. Indeed, several mAbs and nanobodies derived against the S1-RBD, S1-NTD, and S2 of SARS-CoV and MERS-CoV might confer cross-activity against virus SARS-CoV-2 viral entry. 27, 52, [55] [56] [57] [58] [59] It was reported that that SARS-CoV specific human mAbs, s309 60 and CR3022, 52,54 were capable of binding to the SARS-CoV-2 effectively. 61 Accordingly, sera from recovered patients of Covid-19 (as a potential source of nAbs) were used to generate mAbs against SARS-CoV-2. Four of the generated mAbs (31B5, 32D4, P2C-2F6 and P2C-1F11) indicated high neutralizing activity in vitro by efficiently inhibiting ACE2-RBD binding. 25, 46, 62, 63 Alternatively, mAbs 47D11 and n3130 produced from SARS-CoV and SARS-CoV-2 respectively were shown to neutralize SARS-CoV-2 without inhibiting ACE2-RBD binding. 64, 65 In several other recent studies, Abs from convalescent Covid-19 patients (which are correlated with the S1, RBD and S2 regions) were used to treat SARS-CoV-2 infection. 27, 28 Animal models were also used to generate nAbs against SARS-CoV-2.

Structure of SARS-CoV-2 S protein in the pre-fusion conformation. In the top row, the ribbon diagram shows single protomer of SARS-CoV-2 S consists the RBD (green) in the down conformation (closed RBD; left) and surface diagrams show side (centre) and apical (right) views of the structure of SARS-CoV-2 S trimer with three RBD (green and grey) in the down conformation (closed SARS-CoV-2 S trimer). In the bottom row, ribbon diagram shows single protomer of SARS-CoV-2 S consists the RBD (green) in the up conformation (opened RBD; left) and surface diagrams show side (centre) and apical (right) views of the structure of SARS-CoV-2 S trimer with single RBD (green) in the up conformation (opened SARS-CoV-2 S trimer). The structure of the SARS-CoV-2 S protein (PBD ID: 6VSB) 32 Taken together, from findings of resent studies, it might be concluded that S1 and particularly RBD could be considered as the main antigen candidates in vaccine platform formulations to induce virus-specific nAbs to prevent SARS-CoV-2 infection. 78, 79, 81 In Covid-19-induced severe pneumonia, higher levels of nearly similar cytokines/chemokines were correlated to lung injury, indicating that the cytokine storm and exacerbated inflammatory responses were manifested clinically by acute respiratory F I G U R E 3 Immune response to SARS-CoV-2. Dendritic cells as APC present viral antigens to CD4þ T cells and induce the production of IgG, IgM and IgA to prevent viral entrance. Furthermore, cytokine storm starts in severe cases that might be correlated with disease severity. It was shown that antibodies and CD4þ T cells generated in 100% of recovering Covid-19 patients. The CD8þT cells also detected in 70% of recovering COVID patients which secrete perforin and granzyme to kill virally infected cells. It was found CD4þ T-cell responses to S protein, the main target of most vaccine efforts, were robust, and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titres. T cell responses are focused not only on S but also on M, N, and other ORFs. APC, antigen-presenting cell; Covid-19, coronavirus disease 2019; SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2 ARASHKIA ET AL. T cells were generated in around 70% and 100% of Covid-19 patients, respectively. 29 The identified CD4þ T cell responses were strong and associated with the induction of IgG and IgA antibody. Of note, 50% of the total CD4 þ T cell responses were against the S protein, while the specific CD8þ T cells against S protein were also found in most, if not all participants. 29, 91 In another study, 13 out of 14 recovered Covid-19 patients, showed strong correlation between nAb titres and the numbers of virus-specific T cells. 92 Accordingly, results of a study on ten Covid-19 patients with moderate to severe ARDS showed strong and specific CD4þ and CD8þ cells mostly against S protein in 100% and 80% of patients, respectively. 93 These cellular responses were mainly skewed towards Th1, although Th2 and Th17 cytokines were also found. Besides, low levels of specific T-cells were found in 20% of unexposed individuals as a potential indicator of cross-reactive T cell between SARS-CoV-2 and common cold-causing coronaviruses. 93 In parallel, results of a cohort study indicated SARS-CoV-2-specific CD4þ and CD8þ T cells with high cytotoxic activity in the acute phase of the disease 94 (specially S protein specific) implying the role of cellular response in a potential vaccine. It should be noted that, despite reports on safety of SARS-(full) S protein-encoding vaccines in immunized mice or non-human primates [103] [104] [105] [106] and mice immunized passively by anti-S-antibody, 107, 108 but ADE has been observed in cats vaccinated by recombinant vaccinia virus expressing fusogenic S protein. 109 In addition, lung immunopathology and hepatitis have been found in SARS-CoV-challenged animal models after vaccination with SARS-(full) S protein-encoding vaccines, the same as that of whole viral-vaccine. 95, 96, 110, 111 These observations resulted to application of various segments of SARS-CoV-2 S protein including RBD, NTD, S1 and S2 (besides full S fragment). It is reported that the S1 subunit or RBD of S protein induce nAbs without potential of ADE development. 76, 112 

In several previous studies, the full-length S protein was used to 107 Accordingly, the administration of full-length S protein trimer to mice or hamsters was also shown to induce significant protection against homologous virus shedding. 108 Similarly, animal immunization with baculovirus, expressing the full-length and extracellular domain of S protein from the SARS-CoV Urbani strain was shown to induce nAbs against homologous and heterologous pseudoviruses of SARS-CoV. 114 Recently, it was shown that SARS-CoV and MERS-CoV S nanoparticles produced in the baculovirus expression system induce high titres of nAbs against the homologous but not the heterologous virus (i.e., no cross-protection). 115 Currently, several developers use full-length S protein as antigen in various platforms to construct an efficient vaccine candidate against SARS-CoV-2 that are currently in the clinical trial or preclinical phases (Table 1: based on WHO draft landscape of Covid-19 candidate vaccines-28 September 2020). 116 transmembrane anchor that has been stabilized by two consecutive proline substitutions (S-2P) of residues 986 and 987. In preclinical evaluation, BALB/c, C57BL/6 and B6C3F1 mice strains were immunized by two-dose IM injection of 0.01, 0.1 or 1 μg of mRNA-1273.

The antibody titres increased with dose level, and a potent neutral- challenge. [138] [139] [140] It should be noted however that while induction of nAbs against RBD is the primary effector response of the protective immunity, T-cell immune responses that might further contribute to the protection were also found following immunization of mice with the RBD-based subunit vaccines. 33, 140, 141 

Alike RBD, NTD in S protein of some CoVs show receptor-binding activity through binding to sugar moieties. 144, 145 Several studies showed that recombinant NTD protein of MERS-CoV is capable of eliciting sufficient nAbs and cellular immune responses to protect against virus challenge in animal models. 33, 146, 147 Although compared to other regions of S protein (full-length S protein, S1 and RBD), NTD is less immunogenic (i.e.,: eliciting considerably lower antibody titres and cellular immune responses), it might be involved in the binding of specific receptors 144, 145 and thus deserve to be considered as a candidate antigen for vaccine development against Covid-19.

The S1 subunit, which contains both RBD and NTD regions, is responsible for virus binding to the host cell receptor. Prior studies indicated that the S1 subunit can induce strong immune responses and/or protection against viral infection. 58, 148 Immunization of rats via subcutaneous or intranasal routes with a recombinant adenovirus encoding first 490 amino acids of the S1 subunit, elicited strong humoral immune responses that protected the animals against SARS-CoV infection. 149 Similarly, immunization of hDPP4 transgenic mice with MERS-CoV recombinant S1 protein, formulated with MF59 adjuvant, induced nAbs which correlated with protection. 150 A similar study also reported that intramuscular injection of an adjuvant formulated MERS-CoV S1 protein (subunit vaccine) was capable of reducing virus shedding in dromedary camels, while conferring complete protection against the viral challenge in alpaca. 148 Recently, it was shown that subcutaneously immunized mice (either traditional needle injection or intracutaneously by dissolving microneedle arrays

[MNAs]), by a codon-optimized S1 subunit containing integrated (inbuilt) TLR agonist sequences, elicited specific humoral responses which were of higher titres in MNAs delivery. 151 Therefore, the S1 subunit of SARS-CoVs might also have the potential to be considered as the main antigen in different platforms to formulate a vaccine candidate against these viral infections.

To date, the vaccine candidates that use SARS-CoV-2 S1 as the primary antigen are in the preclinical stage and include a protein subunit vaccine platform co-developed by AnyGo Technology (recombinant S1-Fc fusion protein), University of Pittsburgh (microneedle arrays S1 subunit), and Baylor College of Medicine and also a recombinant deactivated rabies virus platform developed by Bharat Biotech/Thomas Jefferson University. 116 

The S2 subunit, which contains an internal membrane fusion peptide (FP) and heptad repeats (HR1 and HR2), is responsible for fusion between the viral and host cell membranes. The S2 subunit which is highly conserved among SARS-CoVs and MERS-CoV is an immunogenic protein. 89, [152] [153] [154] [155] Several studies have reported that HR1

and HR2 domains of S2 can generate broadly nAbs against pseudotyped heterologous SARS-CoVs in vitro. 153, 156, 157 It should be noted however that other regions of S2 domain (residues 681-980) might elicit non-nAbs (as shown in immunized mice). 158 In addition, an S2 peptide sequence (residues 736-761) of MERS-CoV induced S2-specific nAbs in rabbits, 131 although the protective efficacy is yet to be addressed. Recently, linear epitopes of SARS-CoV-2 S2 were mapped and the presence of cross-reactive neutralizing epitopes for other SARS-CoVs were shown, which sounds a more promising role for this protein as main antigen of a vaccine platform. 89 It should be noted however that the FP domain of S2 which is involved in the host cell membrane fusion and viral pathogenicity has also the potential of being used as an antigen of vaccine platforms, either alone or fused with other antigenic fragments (RBD, NTD, HR1 and HR2). To date, a RBD-FP fusion protein that induced strong antibody response in immunized mice was constructed 159 but its protective efficacy remains to be addressed.

The ACE2 cellular receptor binding, S protein of SARS-CoV-2, plays an essential role in viral entry and infection and contains multiple B-

and T-cell epitopes to induce nAbs and long-term protection, suggesting a main candidature for this protein as Vaccine antigen.

Accordingly, three vaccines based on full-length-S antigen including 

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Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

Receptor-binding domain of SARS-CoV spike protein induces long-term protective immunity in an animal model

Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen

Priming with rAAV encoding RBD of SARS-CoV S protein and boosting with RBD-specific peptides for T cell epitopes elevated humoral and cellular immune responses against SARS-CoV infection

Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus S protein elicits neutralizing antibodies: implication for developing SARS vaccines

Intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein induces strong mucosal immune responses and provides long-term protection against SARS-CoV infection

Identification of murine CD8 T cell epitopes in codon-optimized SARS-associated coronavirus spike protein

Concurrent Human Antibody and TH1 type T-cell Responses Elicited by a COVID-19 RNA Vaccine

Phase 1/2 Study to Describe the Safety and Immunogenicity of a COVID-19 RNA Vaccine Candidate (BNT162b1) in Adults 18 to 55 Years of Age: Interim Report

Crystal structure of the S1 subunit N-terminal domain from DcCoV UAE-HKU23 spike protein

Identification of the receptor-binding domain of the spike glycoprotein of human betacoronavirus HKU1

The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

A novel neutralizing monoclonal antibody targeting the N-terminal domain of the MERS-CoV spike protein

Efficacy of an adjuvanted Middle East respiratory syndrome coronavirus spike protein vaccine in dromedary camels and alpacas

Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats

Receptor-binding domain of MERS-CoV with optimal immunogen dosage and immunization interval protects human transgenic mice from MERS-CoV infection

Microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development

Human monoclonal antibodies against highly conserved HR1 and HR2 domains of the SARS-CoV spike protein are more broadly neutralizing

Prospects for a MERS-CoV spike vaccine

The outbreak of SARS-CoV-2 pneumonia calls for viral vaccines

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

Subunit vaccines against emerging pathogenic human coronaviruses

Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor

Elicitation of immunity in mice after immunization with the S2 subunit of the severe acute respiratory syndrome coronavirus

Progress and prospects on vaccine development against SARS-CoV-2. Vaccines

Severe acute respiratory syndrome-coronavirus-2 spike (S) protein based vaccine candidates: State of the art and future prospects

The members of the Rapid Response Team of PII also include Fate- 

The authors declare no conflicting financial or other interests.

All authors contributed to the writing of the manuscript.

Data openly available in a public repository that issues datasets with DOIs.

https://orcid.org/0000-0001-6390-8399Zabihollah Shoja https://orcid.org/0000-0001-5617-5844