Progress in research on the S protein as the target of COVID-19 vaccines

KEYWORDS:

• COVID-19
• vaccines
• S protein
• neutralizing antibodies
• two proline substitutions

The spike protein of SARS-CoV-2 is the first target identified by researchers on COVID-19 vaccines. The locus as a homotrimer in the virus envelope consists of two subunits, namely, S1 and S2. In MERS-CoV, SARS-CoV, and SARS-CoV-2, the receptor-binding domain (RBD) in the S1 subunit binds to host cells, whereas the S2 subunit is needed for viral entry through membrane fusion. The S1 subunits of certain CoVs consist of an N-terminal domain (NTD) and a C-terminal domain (CTD). The NTD is utilized for directed receptor binding to host cells, such as in mouse hepatitis virus, or undirected receptor binding through sugar molecules, such as in Infectious bronchitis virus, transmissible gastroenteritis virus, and bovine coronavirus [1]. Moreover, the NTD is involved in pre – and post-fusion of virus proteins and RNA. By contrast, the RBD region located at the CTD, like the S2 subunit, is composed of several regions, such as a connecting region (CR) and fusion peptide (FP) heptad repeats 1 and 2. Crystal structure modeling proposed a model for the configuration of the SARS-CoV-2 S protein after the identification of host cell receptors. FP constraints are released when S1 and S2 are cleaved. Interactions between human angiotensin-converting enzyme 2 and RBDs contribute to the detachment of the S1 subunit and the refolding process as an extended helical bundle of the S2 subunit, leading to protrusions to FP in the membrane of host cells [2]. In theory, neutralizing antibodies (nAbs) target the S protein during the entry phase of the virus to prevent viral infection at multiple levels. The key target for nAbs and binding in viral receptors is RBD [3,4].

Interestingly, the pioneering work of Xiangyang et al [5]. demonstrated that the 4A8 monoclonal antibody from convalescent Covid-19 patients has a high neutralization potency against both authentic and pseudotyped SARS-CoV-2. By utilizing cryo-electron microscopy, the authors identified the epitope of 4A8 as the NTD of the S protein complex with the S protein with a local resolution of 3.3 angstroms and an overall resolution of 3.1 angstroms [5]. As the antibody binds to the S protein, the 4A8 epitope overlaps the ganglioside binding region, stopping the virus from entering lipid rafts [6]. In addition, spike inactivation by antibody-induced destabilization of the coronavirus’s prefusion structure has been identified as an alternative method of coronavirus neutralization by RBD-targeting antibodies [7]. MRBD-Fc vaccination of human ACE2 transgenic mice acted as a broad-spectrum vaccine and offered adequate protection against SARS-CoV-2 infection [8].

The nAbs targeting NTD have also been observed in MERS-CoV and SARS-CoV-2. This process is another possible option that has implications in vaccine development. Given that the S2 subunit and FP are necessary for membrane fusion in host cells, they may be a second target for nAbs. Moreover, T cells targeting the S protein have been identified in SARS-CoV, MERS-CoV, and SARS-CoV-2. Both CD4 + and CD8 + T cells target S protein epitopes [9]. Four vaccine candidates for COVID-19 based on the adenovirus–vector strategy that analyzes the full length of the S protein have reached phase III clinical trials and exhibited the ability to stimulate helper cell (TH1) responses and nAb production. The first such vaccine, called ChAdOx1 (Table 1), which is based on recombinant chimpanzee adenovirus, was developed in the UK. The second such vaccine was developed in Russia. This vaccine integrates recombinant human Ad5 and Ad26 in a prime–boost vaccine regimen. The third such vaccine was developed in China. This vaccine uses human adenovirus type 5 (Ad5). Preliminary results showed that the ChAdOx1 vaccine induces nAb production immune responses in nonhuman primates (NHPs), and this vaccine reduces viral loads in the respiratory tract and bronchoalveoli and prevents COVID-19 pneumonia [10,11]. A single dose of ChAdOx1 nCoV-19 was used to induce a TH1-biased response characterized by CD4 + T cell secretion of interferon and tumor necrosis factor, as well as antibody generation primarily of IgG1 and IgG3 subclasses. Monofunctional, polyfunctional, and cytotoxic phenotype CD8 + T cells were also induced [11].

Table 1. Progress in research on the S protein as the target of COVID-19 vaccines

Strategy	Two proline substitutions (2P)	Mutation	Company/University	Contrary	T cell responses	Stimulate of Specific IgG	Mice	human	Other animals	Stimulate of neutralizing antibodies nAbs	Clinical trials Phase
S-protein Full-length
(ChAdOx1) Virus vector			University of Oxford & AstraZeneca	United Kingdom	high to TH1 Low to TH2	S-specific	✔	✔		Yes	III
(Ad26 and Ad5) Virus vector			Gamaleya Research Institute	Russia		RBD-specific		✔		Yes	III
(Ad26) Virus vector			CanSino Biological Inc. with Beijing Institute of Biotechnology	China	TH1	RBD-specific		✔		Yes	III
DNA			Inovio Pharmaceuticals	Korea		S1-specific & RBD-specific	✔		guinea pigs	Yes	II
S-protein Full-length + Two proline substitutions (2P)
mRNA	✔	(K986P and V987P) in 2P	BioNTech, with Fosun Pharma and Pfizer	Germany		S1-specific		✔		Yes	III
Protein	✔	(K986P and V987P) in 2P,(R682Q, R683Q and R685Q) in S protein	Novavax	United States	high to TH1 Low to TH2	S-specific	✔	✔	NHPs	Yes	III
(Ad26) Virus-vectored	✔	K986P and V987P) in 2P, (R682S and R685G) in S protein	Janssen Pharmaceutical Companies	United States	high to TH1 Low to TH2	RBD-specific		✔	NHPs	Yes	III
LNP-mRNA	✔	K986P and V987P) in 2P	Moderna	United States	high to TH1 Low to TH2	S-specific	✔	✔	NHPs	Yes	III

The second vaccine was developed in Russia via integrating the recombinant human Ad5 and Ad26 in a prime–boost vaccine regimen. The heterologous rAd26 and rAd5 vector-based COVID-19 vaccine has a good protection profile and induced high humoral and cellular immune responses in participants at day 28 by upregulating the median cell proliferation of CD4+ and CD8 + T cells in both frozen and lyophilized formulations, according to phase 1/2 studies conducted at two hospitals in Russia. In this regard, at day 42, the RBD-specific IgG titers were 14,703 in the frozen formulation and 11,143 in the lyophilized formulation, with a seroconversion rate of 100%. The nAbs were 4925 in the frozen formulation and 4595 in the lyophilized formulation [10]. The third vaccine was developed in China as cooperation of CanSino Biological Inc. with Beijing Institute of Biotechnology, China, and introduced into phase III clinical trials. This vaccine uses human adenovirus type 5 (Ad5) to induce RBD-specific IgG and nAbs in humans, as well as TH1 cell responses [12]. The fourth vaccine was developed by Janssen Vaccines Leiden in the Netherlands, and it is known as Janssen-Ad26.COV2.S. Substantially, the multicenter placebo-controlled phase 1–2a trials showed that the CD4 + T-cell responses on day 15 were detected in two cohort studies with clear skewing toward TH1 cell responses. Overall, CD8 + T-cell responses were strong but weak in cohort 3 [13].

DNA vaccine strategies based on the expression of the S protein are undergoing phase II clinical trials in tandem with the adenovirus–vector strategy. DNA vaccines stimulate T cells and nAbs in both pigs and mice [14]. Furthermore, AG0302-COVID19 is a DNA-based vaccine funded by Anges, Inc. in Japan that recently completed phase I/II trials, but the findings of these studies have not yet been released. In rat experiments, AG0302-COVID19 induced antibodies that inhibited SARSCoV-2 S protein binding via S2 and RBD recognition [15].

Another vaccine strategy is based on the trimeric spike protein provided by Clover Biopharmaceuticals AUS Pty Ltd. A disulfide-bonded homotrimer form of the CTD type Iα collagen from humans combines with the S protein from the virus to increase the vaccine’s stability and antigen yield. This vaccine can stimulate nAbs because it is structurally similar to the native form of the S protein. Regarding this approach, a double-blind, randomized, controlled study was conducted to assess the effectiveness of the CpG 1018/Alum-adjuvated recombinant SARS-CoV-2 trimeric SCB-2019 vaccine against COVID-19 and SARS-CoV-2 infection, immunogenicity, reactogenicity, and protection. This study’s findings were significant in that they demonstrated strong neutralizing antibody responses, a TH1-biased cellular immune response, and an adequate safety profile. Thus, the recommended candidates for the phase 2/3 trial are 9 μg of SCB-2019 adjuvanted with AS03 and 30 μg of SCB-2019 adjuvanted with CpG/Alum [16].

A new strategy similar to the HIV vaccine strategy has been developed and has reached phase I clinical trials. This strategy combines S homotrimers either with CpG 1018 (a Toll-like receptor 9 agonist adjuvant) or AS03 (a squalene-based adjuvant) to produce high immune responses by TH1 cells and nAbs in NHPs and mice [17]. This vaccine protects NHPs from SARS-CoV-2 infection and minimizes viral loads in the lungs. However, when formed as a recombinant protein, the S protein becomes unstable and likely transfigures from its pre-fusion conformation into its post-fusion conformation, leading to the removal of the S1 subunit. Given that the S1 subunit consists of the epitopes of RBD, the S protein should be stabilized in its pre-fusion structure. The heptad repeat 1 (HR1) and two proline substitutions (2P) can maintain the S protein’s pre-fusion conformation in human coronavirus, MERS-CoV, and SARS-CoV [18].

S-2P is antigenically optimal because it stimulates more nAbs than the native S protein in mice. This strategy was adopted against SARS and is now utilized in some COVID-19 vaccines. Mutant variations of 2P K986P and V987P are used in SARS-CoV-2 S-2P mRNA vaccines by BioNTech/Pfizer, Moderna, Novavax, and Janssen Pharmaceutical Companies. Adding an extra mutation in S protein increases the stability of the pre-fusion conformation and now these mutations being evaluated in phase III clinical trials by animal lab models. Some studies reported that the S-2P expression in the Ad26 vaccine from Janssen induces high levels of nAbs and is safer than the other native S protein vaccines in NHPs [19,20]. Furthermore, mRNA vaccines from BioNTech/Pfizer and Moderna provide high levels of nAbs and significant T cell responses in convalescent patients. Consequently, an efficient way of enhancing the effectiveness of S protein vaccines is by stabilizing the pre-fusion conformation of the S protein. Data from both BioNTech/Pfizer and Moderna confirmed that mRNA vaccines are about 90% effective in preventing COVID-19 infection [21]. These vaccines have reached phase III clinical trials. The BioNTech/Pfizer vaccine has been approved by the FDA for use in many countries. Hsieh et al. developed a new form of the S protein called HexaPro, which consists of six proline mutation loci in CR, FP, and HR1 that lead to about a tenfold increase in both folding and stabilization of the S protein’s pre-fusion conformation. This strategy opens a new area in antigen design and the development of new vaccines [22]. Collectively, several promising vaccines have completed the final stages of large-road clinical trials for vaccine safety and protection effectiveness, with recent reports from BioNTech/Pfizer and Moderna/NIAID citing safety and very high rate of protection efficacy for their leading mRNA vaccine candidates. It is worth noting that comparing vaccines is currently problematic due to the lack of standardized assays for neutralization and challenge tests.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Acknowledgments

This work was supported by Shandong University postdoctoral fellowship to Mohnad Abdalla.

Additional information

Funding

This work was supported by the Shandong Provincial Key Research and Development Program (Major Scientific and Technological Innovation Project) (2019JZZY021013)，Shandong Provincial Key Research and Development Program (2019GSF108080), Funds for Youth Interdisciplinary and Innovation Research Groups of Shandong University (2020QNQT003).