SARS-CoV-2 virus: Vaccines in development

Abstract

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, is an unprecedented challenge to humanity. Global herd immunity may be necessary before resumption of normal economic and societal activities. Since the beginning of the outbreak, the development of COVID-19 vaccines has proceeded at record speed using nearly all available platforms or strategies to maximize vaccine success. A total of 42 vaccine candidates have now entered clinical trials and encouraging data from several vaccine candidates in phase 1 or 2 clinical trials have been reported. In this review, we examine current COVID-19 vaccine candidates, discuss their strengths and weaknesses, summarize published clinical data and analyze future challenges.

1. Introduction

At the end of December 2019, a novel coronavirus designated as SARS-CoV-2 emerged and caused an outbreak of unusual virus pneumonia [1], [2], [3], [4], [5]. Because of its extraordinary transmissibility, coronavirus disease 2019, caused by this novel virus, rapidly caused a global pandemic and brought the world to a near standstill [6], [7], [8], [9]. By late October 2020, the virus had accounted for more than 40 million laboratory-confirmed infections and 1 million deaths in 218 countries and territories, and still counting. In the absence of a vaccine, physical-distancing, face masks and other transmission-mitigation strategies have been implemented to manage COVID-19 [10,11]. While these strategies reduce the risk of infection they do not provide immunity to SARS-CoV-2 and individuals remain susceptible to the virus. Normal economic and social activities will not be resumed until global herd immunity is achieved. Due to the emergence of re-infection and the fact that even a 1% mortality rate to COVID-19 will kill millions of people globally, the dependence of humankind immunity on infection is unacceptable [12], [13], [14]. Therefore, safe and effective vaccines against COVID-19 are desperately needed.

As SARS-CoV-2 is a new virus, knowledge of what constitutes a safe and immunologically effective vaccine strategy is limited [15,16]. Nevertheless, the global pandemic required quick action and development of vaccines at record speed. Since the beginning of the pandemic, various vaccine platforms and strategies including live attenuated virus, inactivated virus, protein subunit, virus-vectored, mRNA and DNA vaccines, have been developed in parallel to maximize the potential success rate [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. These efforts are encouraging and fruitful. According to the World Health Organization (WHO), there are currently 10 vaccine candidates being evaluated in phase 3 clinical trials, 34 in phase 1 or 2 clinical trials, and 154 in pre-clinical development. During COVID-19 vaccine development, a new pandemic vaccine development paradigm has been built and vaccine development time has been compressed from the typical 10–15 years to only 1–2 years. Results from phase 1 or 2 clinical trials of eight COVID-19 vaccine candidates have already been reported [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]. This review outlines strengths and shortfalls of every COVID-19 vaccine development platform, summarizes published clinical data, and analyzes the challenges ahead.

2. Target antigen for COVID-19 vaccines

SARS-CoV-2 is an enveloped, positive-sense RNA virus in the genus Betacoronavirus of the family Coronaviridae [43]. The virion envelope consists of membrane (M) and envelope (E) proteins, coated with a “crown”-like trimeric spike (S) protein. Nucleocapsid (N) proteins bound to the RNA genome constitute the virion core [44], [45], [46]. Similar to other human coronaviruses such as SARS-CoV, SARS-CoV-2 uses an S protein for entry into host cells [47], [48], [49]. The S protein can be functionally categorized into S1 and S2 subunits, which are separated by a protease cleavage site [48,50,51]. First, the receptor-binding domain (RBD) at the C terminus of the S1 subunit engages human angiotensin-converting enzyme 2 (hACE2) as the receptor, which subsequently causes fusion between the viral envelope and the host cell membrane through the S2 subunit [8,45,[52], [53], [54]]. RBD, S1 and the S protein (full-length or ectodomain) are capable of eliciting highly potent neutralizing antibodies and cellular immunity and have been widely selected as promising antigens for COVID-19 vaccine development [25,29,31,40,55,56].

The structures of the RBD alone, RBD-hACE2 and RBD-monoclonal antibody have been determined at high resolution by many research groups [57], [58], [59]. SARS-CoV-2 RBD contains two structural domains [58]. One is the conserved core subdomain with five antiparallel β strands, and the other is the external subdomain which is dominated by a disulfide bond-stabilized flexible loop and responsible for the recognition of hACE2 (Fig. 1 ) [57,58]. The Cryo-EM structure of SARS-CoV-2 S trimer revealed that two dominant distinct conformational states exist, one is closed S trimer, and the other is partially open S trimer with one of the three RBDs rotated up in a receptor-accessible conformation (Fig. 2 ) [57,60]. These findings provided important structural information about the vaccine antigens [43,52,[61], [62], [63]]. However, in addition to the S protein, other proteins, such as the N protein, M protein and non-structural proteins may be potential vaccine antigens and act by inducing a more balanced immune response between humoral and cellular immunity.

Fig. 1.

The structure of SARS-CoV-2 RBD (PDB:6LZG) [58].

Fig. 2.

The two distinct conformational states of SARS-CoV-2 S trimer [57]. (a) Closed SARS-CoV-2 S trimer with all RBDs closed (PDB:6VXX). (b) Partially open SARS-CoV-2 S trimer with one RBD at the trimer apex open (PDB:6VYB).

3. COVID-19 vaccine candidates in development

3.1. Live attenuated vaccines

Live attenuated vaccines are based on an attenuated strain of virus that can induce a host immune response by limited replications in vivo but not lead to disease. The attenuation strategies generally include adaption of viruses to unfavorable conditions such as low temperature or rational gene modifications involving codon de-optimization and deletion of certain genes associated with replication or eluding human immune recognition [64,65]. An advantage of such vaccines is that they mimic natural infections and can stimulate potent cellular response aside of induction of virus neutralizing antibodies. The disadvantage to these vaccines is the safety concern about virus reverse mutations in vaccinated individuals. To date, only three SARS-CoV-2 live attenuated vaccine candidates have reached pre-clinical stages, including one (developed by Codagenix and the Serum Institute of India) by using the CodaVax technology platform which attenuates the virus through codon de-optimization.

3.2. Inactivated vaccines

Purified inactivated vaccines are widely used for prevention of diseases caused by viruses such as the influenza virus and the poliovirus [66], [67], [68]. Selection for a seed virus strain, characterized by highly efficient proliferation and high genetic stability, is the first step for developing SARS-CoV-2 inactivated vaccines. The selected strain is then propagated in cell cultures, usually vero cells followed by inactivation using chemicals such as β-propiolactone. After optimized purification steps including chromatography, pure inactivated vaccine preparations are obtained. Because of the whole virus being presented to the immune system, an advantage of inactivated vaccines is that the elicited immune response will target not only the S antigen but also other structural or non-structural proteins including the M, E and N proteins. The inclusion of other viral antigens might induce a more balanced response involving both humoral and cellular immunity. However, pulmonary immunopathology induced by SARS-CoV vaccine candidates, either directly mediated by a Th2 immune response or as a result of antibody-dependent enhancement (ADE) [69], [70], [71], [72], warrants a careful investigation of the safety of these vaccines against SARS-CoV-2. Certain epitopes, which readily induce high-binding but low-neutralizing antibodies, are prone to elicit an ADE effect. Thus, the inactivated vaccine type may have a high ADE risk because of the maximal epitopes included. In addition, the requirement for biosafety level 3 (BSL3) production facilities during SARS-CoV-2 propagation might be a limitation to inactivated vaccine production.

Beijing Institute of Biological Products Company Limited, together with China CDC and Peking Union Medical College, developed a SARS-CoV-2 inactivated vaccine with alum as adjuvant (BBIBP-CorV) [33]. A randomized, double blinded placebo-controlled phase 1/2 trial of this vaccine has been reported (ChiCTR2000032459). A total of 192 healthy adults (18–80 years) and 448 adults (18–59 years) were enrolled in phase 1 and 2 trials, respectively. In phase 1, subjects were separated into two age groups (18–59 years and ≥ 60 years) and the developers tested three doses, 2, 5, and 8 μg of BBIBP-CorV in a prime-boost vaccination regimen within a 4-week internal. In phase 2, a single-dose schedule of 8 μg and a two-dose schedule of 4 μg with internals of 2, 3, or 4 weeks were investigated. In both trials, all of the adverse reactions were mild or moderate in severity and no serious adverse events were reported. The phase 1 trial indicated that neutralizing antibody titers in adults receiving 4 μg doses at two weeks post boost reached 211.2 in the young group and 131.5 in the elderly group, respectively. The lower neutralizing antibody titer in the elderly group indicates the need for an increased dose and/or more potent adjuvant. In the phase 2 trial, two-dose immunization of the 4 μg vaccine with internals of 3 or 4 weeks achieved higher neutralizing antibody titers than the single 8 μg dose or 4 μg dose with a two-week internal. This indicated the benefit of increasing the internal time between prime and boost injections. During the revision of the manuscript, BBIBP-CorV has been reported of a protection efficacy of 78.4% and been approved for use in China.

Interim evaluation of randomized, double-blind, placebo-controlled, phase 1 and 2 clinical trials of an alum-adjuvanted inactivated vaccine candidate (developed by Wuhan Institute of Biological Products and Sinopharm) was also reported (ChiCTR2000031809) [34]. The phase 1 trial with 96 adults (aged 18–59 years) tested three doses, 2.5, 5, and 10 μg with three injections, 4 weeks apart. The phase 2 trial used 224 adults (aged 18–59 years) and a 5 μg dose in a prime-boost vaccination regimen with 2- and 3-week internals. The vaccine was well tolerated and showed excellent safety in both trials. In the phase 1 trial, the geometric mean titers of neutralizing antibodies at 2 weeks post 3 injections reached 316 (low-dose group), 206 (medium-dose group), and 297 (high-dose group), respectively. In the phase 2 trial, similar to the results of BBIBP-CorV, neutralizing titers in the 3-week internal group (247) were higher than those in the 2-week internal group (121).

Another inactivated vaccine candidate with alum as the adjuvant (CoronaVac, also designated as PiCoVacc, developed by Sinovac and the Chinese Academy of Medical Sciences), was the first COVID-19 vaccine candidate evaluated in the rhesus macaque model [17]. The developer found that three immunizations within the one-week internal at 3 μg and 6 μg doses could induce moderate neutralizing antibody titers and protect macaques from virus infection in the lower respiratory tract following SARS-CoV-2 challenge without evident antibody-dependent enhancement of infection. The three vaccine candidates mentioned above are currently being evaluated in phase 3 clinical trials. Other inactivated vaccine candidates involving one Kazakh, one Indian and one Chinese candidate are currently in phase 1 or 2 clinical trials (Table 1 ).

Table 1.

The development of vaccine candidates in clinical trial.

Platform	Vaccine	Developer	Location	Antigen	Adjuvant	Route	Number of dose	Phase
Inactivated virus	CoronaVac	Sinovac	China	Whole-virus inactivated	Alum	IM	2	Phase 3 NCT04456595
	BBIBP-CorV	Beijing Institute of Biological Products Company Limited	China	Whole-virus inactivated	Alum	IM	2	Phase 3 ChiCTR2000034780 NCT04560881
	Inactivated vaccine	Wuhan Institute of Biological Products	China	Whole-virus inactivated	Alum	IM	2	Phase 3 ChiCTR2000034780 ChiCTR2000039000
	BBV152	Bharat Biotech	India	Whole-virus inactivated	Aluminum hydroxide gel (Algel) or a novel TLR7/8 agonist adsorbed Algel (Algel-IMDG)	IM	2	Phase 1/2 NCT04471519 CTRI/2020/09/027674
	QazCovid-in®	Research Institute for Biological Safety Problems	Rep of Kazakhstan	Whole-virus inactivated	Not available	IM	2	Phase 1/2 NCT04530357
	Inactivated vaccine	Institute of Medical Biology, Chinese Academy of Medical Sciences	China	Whole-virus inactivated	Not available	IM	2	Phase 1 NCT04412538
	Inactivated vaccine	Beijing Minhai Biotechnology	China	Whole-virus inactivated	Not available	IM	1, 2 or 3	Phase 1 ChiCTR2000038804
Platform	Vaccine	Developer	Location	Antigen	Adjuvant	Route	Number of dose	Phase
Protein subunit	ZF2001	Anhui Zhifei Longcom Biopharmaceutical	China	Dimeric RBD (CHO cells)	Alum	IM	2 or 3	Phase 2 NCT04466085
	KBP-COVID-19	Kentucky Bioprocessing Inc	America	RBD (tobbaco plants)	Not available	IM	2	Phase 1/2 NCT04473690
	Soberana 01	Instituto Finlay de Vacunas	Cuba	RBD	Adjuvant	IM	2	Phase 1 IFV/COR/04
	COVID-19 recombinant vaccine (Sf9)	West China Hospital, Sichuan University	China	RBD (insect cells)	Alum	IM	2	Phase 1 ChiCTR2000037518
	UB-612	COVAXX, United Biomedical	America	RBD fused to Fc domain of human IgG1, combined with SARS-CoV-2 peptide pool	AdjuPhos® (CpG1 plus alum)	IM	2	Phase 1 NCT04545749
	COVAX19	Vaxine Pty Ltd, Medytox	Australia	S (insect cells)	Advax-SM	IM	2	Phase 1 NCT04453852
	Sclamp (COVID-19) Vaccine	University of Queensland	Australia	Molecular clamp stabilized S (mammalian cells)	MF59	IM	2	Phase 1 ACTRN12620000674932p
Platform	Vaccine	Developer	Location	Antigen	Adjuvant	Route	Number of dose	Phase
Protein subunit	NVX-CoV2373	Novavax	America	S with cleavage site deleted and two stabilizing proline mutations (insect cells)	Saponin-containing Matrix-M	IM	2	Phase 3 2020-004123-16
	Recombinant protein vaccine	Sanofi Pasteur, GSK	France	S (insect cells)	No adjuvant or AS03	IM	2	Phase 1/2 NCT04537208
	SCB-2019	Clover Biopharmaceuticals , GSK, Dynavax	China	Trimeric S protein (mammalian cells)	No adjuvant, AS03 or CpG1018 plus Alum	IM	2	Phase 1 NCT04405908
	MVC-COV1901	Medigen Vaccine Biologics Corporation, NIAID, Dynavax	China Taiwan	S with cleavage site deleted and two stabilizing proline mutations (insect cells)	CpG 1018 plus alum	IM	2	Phase 1 NCT04487210
	HBsAg VLPs	SpyBiotech, Serum Institute of India	India	RBD-HBsAg	Not available	IM	2	Phase 1/2 ACTRN12620000817943
	CoVL	Medicago	Canada	Coronavirus-like particle	No adjuvant, AS03 or CpG1018	IM	2	Phase 1 NCT04450004
Platform	Vaccine	Developer	Location	Antigen	Adjuvant	Route	Number of dose	Phase
Virus vectored	ChAdOxnCoV-19	University of Oxford, AstraZeneca	England	Replication-deficient chimpanzee Ad-based S	No	IM	1	Phase 3 ISRCTN89951424 NCT04516746 NCT04540393 CTRI/2020/08/02717
	Recombinant Ad5 vectored COVID-19 vaccine	CanSino, Beijing institute of Biotechnology	China	Replication-deficient human Ad5-based S	No	IM	1	Phase 3 NCT04526990 NCT04540419
	Gam-COVID-Vac	Gamaleya Research Institute	Russia	Replication-deficient human Ad26 (priming), Ad5 (boost)-based S	No	IM	2	Phase 3 NCT04530396 NCT04564716
	Ad26.COV2.S1	Janssen Pharmaceutical Companies	America	Replication-deficient human Ad26-based S1	No	IM	2	Phase 3 NCT04505722
	GRAd-COV2	ReiThera, LEUKOCARE, Univercells	Italy, Germany, Belgium	Replication-deficient Simian Ad-based S	No	IM	1	Phase 1 NCT04528641
	VXA-COV2-1	Vaxart	America	Replication-deficient human Ad5-based	No	Oral	2	Phase 1 NCT04563702
Platform	Vaccine	Developer	Location	Antigen	Adjuvant	Route	Number of dose	Phase
Virus vectored	MVA-SARS-2-S	Ludwig-Maximilians, University of Munich	Germany	Replication-defective MVA-based S	No	IM	2	Phase 1 NCT04569383
	V590-001	Merck Sharp & Dohme, IAVI	Germany	Replication-competent VSV-based S	No	IM	1	Phase 1 NCT04569786
	TMV-083	Institute Pasteur, Themis, University of Pittsburg CVR, Merck Sharp & Dohme	France	Replication-competent measles-vector based	No	IM	1 or 2	Phase 1 NCT04497298
	DelNS1-2019-nCoV-RBD- OPT1	Beijing Wantai Biological Pharmacy, Xiamen University	China	Replication-competent intranasal flu-based-RBD	No	IM	1	Phase 1 ChiCTR2000037782
	hAd5-S-Fusion+N-ETSD	ImmunityBio, NantKwest	America	Replication-deficient human Ad5-based S fusion protein and nucleocapsid with an enhanced T-cell stimulation domain	No	SC	2	Phase 1 NCT04591717
	Ad5-nCoV	CanSino Biological Inc	China	Replication-deficient human Ad5-based	No	IM or mucosal	2	Phase 1 NCT04552366
Platform	Vaccine	Developer	Location	Antigen	Adjuvant	Route	Number of dose	Phase
mRNA	mRNA-1273	Moderna, NIAID	America	S-2P	No	IM	2	Phase 3 NCT04470427
	BNT1626b1, BNT1626b2	BioNTech, Fosun Pharma, Pfizer	Germany	Trimeric RBD, S-2P	No	IM	2	Phase 3 NCT04368728
	CVnCOV	CurVac	Germany	Not available	No	IM	2	Phase 1 NCT04449276
	ARCT-021	Arcturus/Duke-NUS	America	Self-replicating mRNA that encodes for the prefusion spike protein	No	IM	Not available	Phase 1/2 NCT04480957
	LNP-nCoVsaRNA	Imperial College London	England	S	No	IM	2	Phase 1 SRCTN17072692
	ARCoV	Abogen	China	RBD	No	IM	2	Phase 1 ChiCTR2000034112
DNA	INO-4800	Inovio Pharmaceuticals, International Vaccine Institute	America	S	No	ID	2	Phase 1/2 NCT04447781 NCT04336410
Platform	Vaccine	Developer	Location	Antigen	Adjuvant	Route	Number of dose	Phase
DNA	DNA vaccine	Cadila Healthcare Limited	India	DNA plasmid vaccine	No	ID	3	Phase 1/2 CTRI/2020/07/026352
	GX-19	Genexine Consortium	Korea	Not available	No	IM	2	Phase 1/2 NCT04445389
	AG0301-COVID19	Osaka University, AnGes, Takara Bio	Japan	Not available	Yes	IM	2	Phase 1/2 NCT04463472 NCT04527081
Peptides	EpiVacCorona	FBRI SRC VB VECTOR, Rospotrebnadzor, Koltsovo	Russia	SARS-CoV-2 pepetides conjugated a carrier protein	Alum	IM	2	Phase 1 NCT04527575
	pVAC-SARS-CoV-2	University Hospital Tuebingen	Germany	SARS-CoV-2 pepetide cocktail based on human HLA II binding screening	ISA 51 VG plus CpG	SC	1	Phase 1 NCT04546841

3.3. Protein subunit vaccines

Protein subunit vaccines are a popular platform to develop vaccines against infectious viruses such as influenza virus, hepatitis B and varicella-zoster virus [66]. When combined with an adjuvant, protein subunit vaccines usually have good safety and immunogenicity profiles. The great amount of production experience gained from currently licensed vaccines is also an advantage [55]. SARS-CoV-2 protein subunit vaccines can be classified into three categories, RBD-based vaccines, S-based vaccines and virus-like particle (VLP) vaccines. The antigen expression systems involve yeast cells, mammalian cells, insect cells and plants. Depending on the expression systems, glycosylation modifications vary and may influence the immune response [73]. As a relatively small protein, RBD faces the challenge of low immunogenicity. Strategies for improving its immunogenicity may be needed. A dimeric form of RBD has been reported to significantly enhance neutralizing antibody response [55]. Recombinant New Coronavirus Vaccine (CHO Cell) based on the dimeric RBD was developed by our team in collaboration with Anhui Zhifei Longcom Biopharmaceutical. This vaccine has completed the phase 2 clinical trial with excellent safety profiles and immunogenicity, and is scheduled to begin a phase 3 trial. In comparison to RBD, wild-type S is metastable and difficult to manufacture on a large scale in vitro. This may impact vaccine yields in the future [61]. Modifications of the deletion of the polybasic cleavage sites and inclusion of two (S-2P) or six proline substitutions can stabilize the S protein, and significantly improve its yields and the immune response generated [74]. Several vaccine candidates including two protein subunit candidates (NVX-CoV2373 and MVC-COV1901) selected the S-2P mutant as the antigens [35]. There are currently 15 protein subunit vaccine candidates in clinical trials and many more in pre-clinical development (Table 1).

Of these, NVX-CoV2373 (developed by Novavax) is a recombinant nanoparticle vaccine, constructed from the full-length S-2P antigen (mentioned above) with a saponin-containing Matrix-M adjuvant. Data from a randomized, placebo-controlled, phase 1 trial in 131 adults (aged 18–55 years) using this vaccine candidate has been published (NCT04368988) [35]. The trial evaluated two doses of this vaccine (5 and 25 μg) with two intramuscular injections, 3 weeks apart. There were no severe adverse events reported, and the geometric mean neutralizing antibody IC_>99% titers at 2 weeks post boost approached 3906 (5 μg group) and 3305 (25 μg group), respectively. Both of these exceeded the levels observed in symptomatic COVID-19 patients (983). In addition, the Matrix-M adjuvant could skew the immune response toward the Th1 phenotype. This vaccine candidate is currently being evaluated in phase 3 clinical trials (Table 1).

3.4. Virus-vectored vaccines

Replication-deficient virus vectors are increasingly being explored as antigen carriers for developing vaccines against infectious diseases [75]. Such vectors are typically disabled from replication by deletions of certain parts of the virus genome and they are engineered to express the targeted antigen protein, in this case SARS-CoV-2 RBD or S protein. Human and chimpanzee adenoviruses (AdV) are the most popular choices in these approaches. The major advantages of these platforms include safety profiles, considerable experience of scalable manufacturing of some of these vectors, and potent stimulation of both humoral and cellular immunity. A disadvantage is that pre-existing vector immunity in humans could weaken vaccine-elicited immune responses by neutralizing partial vectors. The use of rare serotypes (e.g. chimpanzee adenoviruses) in human populations can circumvent this problem. In prime-boost immunization regimens, boosting with one different vector to priming can also escape impact of priming-induced neutralizing antibodies. ChAdOxnCoV-19 (chimpanzee adenovirus, developed by the University of Oxford and AstraZeneca), a recombinant Ad5 vectored COVID-19 vaccine (Ad5, developed by CanSino and Beijing Institute of Biotechnology), Gam-COVID-Vac (Ad26 priming and Ad5 boost, developed by Gamaleya Research Institute) and Ad26.COV2.S (Ad26, developed by Janssen) are currently in phase 3 clinical trials and eight other virus-vectored COVID-19 vaccine candidates have also entered phase 1 or 2 clinical trials (Table 1).

A phase 1/2, single-blind, randomized controlled trial of S-encoding ChAdOx1 nCoV-19 vaccine in 1077 participants (age of 18–55 years), with licensed MenACWY vaccine (a meningitis vaccine) as a placebo, has been reported (NCT04324606) [76]. The majority of participants received one single injection of ChAdOx1 nCoV-19 at a dose of 5 × 10¹⁰ viral particles or the placebo. Ten individuals were immunized in a prime-boost regimen with a 4-week internal. Although no serious adverse events related to ChAdOx1 nCoV-19 were reported, local and systemic reactions were more common in the SARS-CoV-2 vaccine group than those in the meningitis vaccine group, indicating a worse safety profile. Neutralizing antibody titers from a subgroup of 35 participants were measured using three live SARS-CoV-2 neutralizing assays (a plague reduction neutralization assay, PRNT₅₀; a microneutralisation assay, MN₅₀; and CPE-based neutralizing assay, IC₁₀₀), and the medium titters reached 218, 51, and among 4–16 range, respectively. The boost dose increased the titters in the latter two assays to 136 (MN₅₀) and 29 (IC₁₀₀), respectively. Interferon-γ ELISpot responses peaked at 856 spot-forming cells per million peripheral blood mononuclear cells (PBMC) at 2 weeks post vaccination with a background of 50–100 spot-forming cells per million PBMCs, indicating that a cellular immunity was induced. During the revision of the manuscript, ChAdOx1 nCoV-19 (brand name: AZD1222) has been approved for emergency use in England.

CanSino published the first COVID-19 clinical trial results from the phase 1 trial with their recombinant Ad5 vectored COVID-19 vaccine, expressing wild-type full length S protein [36]. Data from a randomized, double-blind, placebo-controlled, phase 2 trial in 508 adults (aged 18 and older) was also reported (NCT04341389)[37]. In the phase 2 trial two doses were tested, 5 × 10⁹ and 1 × 10¹¹ virus particles, each as one shot. Results showed that 9% of participants in the high-dose group and 1% in the low-dose group reported grade 3 adverse reactions (mostly fever). This indicated that the vaccine was relatively reactogenic, especially when delivered at a high dose. SARS-CoV-2 neutralizing antibody titers approached 18.3 in the low-dose group and 19.5 in the high-dose group, respectively, representing low immunogenicity. High pre-existing anti-Ad5 immunity and increasing patient age were found to negatively impact the neutralizing antibody response. Moreover, both doses induced cellular immunity as evidenced in an interferon-γ ELISpot assay.

3.5. mRNA vaccines

mRNA is a relatively new vaccine development platform. Following antigen-encoding mRNA entering cell cytoplasm, antigen is produced by vaccinees’ cells and immune responses are elicited [43]. In contrast to conventional vaccines that require antigen production in eggs or cell culture, mRNA vaccines usually have substantial advantages of versatility, rapid development and potent immunogenicity, especially when delivered by lipid nanoparticles [77], [78], [79], [80], [81], [82], [83]. Many mRNA vaccine candidates against infectious diseases or cancer have shown great promise in clinical trials in recent years [78]. A number of COVID-19 mRNA vaccine candidates have also been developed and produced promising results in preclinical studies [[26], [27], [28],43,84]. BNT162b2 (a candidate of BioNTech in collaboration with Pfizer) and mRNA-1273 (a candidate of Moderna) are among the most advanced candidates in clinical trials (Table 1). Several other mRNA vaccine candidates including one developed by CurVac and one by the Chinese Liberation Army are also in phase 1 or 2 clinical trials (Table 1). However, given that there has been no previous mRNA vaccine licensed for market, some issues might be encountered about large-scale production and long-term storage stability of the vaccine. mRNA-1273 is an mRNA vaccine encapsulated in lipid nanoparticles (LNPs) that encodes the full-length spike protein with two stabilizing proline mutations (S-2P). Moderna conducted the first COVID-19 phase 1 clinical trial with this vaccine in 45 healthy adults, 18–55 years of age, and published its interim results (NCT04283461) [40]. Three doses of 25 μg, 100 μg, and 250 μg in a prime-boost immunization regimen with 4 weeks separation were evaluated. Systemic adverse events were more common following the second vaccination and 0%, 40% and 57% of the participants in the dose-escalation groups, respectively, had fever after the boost vaccination, whereas none of participants reported fever after the prime dose. As to immunogenicity, authentic virus-neutralizing activity was tittered as PRNT₈₀ and the authors found that the geometric mean PRNT₈₀ titers reached 339.7 in the 25-μg group and 654.3 in the 100-μg group, respectively. Both were within the range of convalescent serum specimens. CD4⁺ T cell responses with a strong Th1 bias were elicited by the vaccine. This vaccine candidate is now in the phase 3 clinical trial with the 100 μg dose in adults and elderly individuals.

BioNTech, together with Pfizer, has published safety and immunogenicity data of BNT1626b1, a LNPs-formulated mRNA encoding SARS-CoV-2 RBD fused with a T4 fibritin trimerisation domain, from a placebo-controlled, observer-blinded Phase 1/2 trial (NCT04368728) [41]. The trial in 45 healthy adults (aged 18–55 years) was designed to assess three doses (10, 30 and 100 μg) in a prime-boost immunization regimen with a 3-week internal. Systemic adverse events increased with dose escalation and, similar to mRNA-1273, adverse events also increased in participants after the boost dose in comparison with those following the first dose. SARS-CoV-2 neutralizing geometric mean NT₅₀ titers in sera at 2 weeks post boost reached 180 in the 10 μg group and 437 in the 30 μg group, respectively, compared to 94 for a panel of COVID-19 convalescent human sera. The developers recently presented additional data from a USA clinical trial with a direct comparison between BNT1626b1 and BNT1626b2, which is similar to BNT1626b1 but encodes S-2P (NCT04368728) [42]. Although the neutralizing antibody titers elicited by the two vaccine candidates were comparable, BNT1626b2 showed less systemic reactogenicity, particularly in elderly adults, and it was selected to enter a phase 3 study.

During the revision of the manuscript, both mRNA-1273 and BNT1626b2 have been approved for emergency use based on primary analysis of phase 3 clinical data, which demonstrated their acceptable safety profiles and protection efficacies of 94.1% and 95%, respectively.

3.6. DNA vaccines

DNA vaccines are based on plasmids that contain a transgene of interest to encode the target antigen in vaccinees’ cells. The huge advantages of DNA vaccines include versatility, rapid development, ease of large-scale manufacturing, and high stability [31,85]. However, unlike mRNA vaccines, DNA vaccines need to be delivered into cell nuclei where transcription occurs to express the antigen. There is a potential safety risk of integration into the host genome, which may lead to insertion mutations. Delivery devices, such as electroporators, may be required, which could limit vaccine use. Moreover, DNA vaccines usually show low immunogenicity and need repeated administrations [85]. There are four COVID-19 DNA vaccine candidates in phase 1 or 2 clinical trials (Table 1). Of those, the S-encoding INO-4800 vaccine candidate (developed by US Inovio Pharmaceuticals) can induce a protective immunity in rhesus masques after 2 injections.

4. Conclusion and perspective

The lengthy COVID-19 global pandemic has stimulated a frenetic search for a safe and efficacious vaccine. The global efforts in research and development for COVID-19 vaccines are unprecedented. Since the outbreak began, a handful of vaccine candidates have entered phase 1, 2 and 3 clinical trials within a period of only 6 months. According to published data from several candidates in phase 1, 2 or 1/2 trials, inactivated vaccine and protein subunit vaccines are well tolerated, the mRNA vaccine shows an increased reactogenicity following the second injection, and the Ad5 vectored vaccine seems to perform the worst. As clinical trials have used different assays and readouts (ID₅₀, ID₈₀, ID₉₉ and ID₁₀₀) to determine neutralizing antibody titers, it is difficult to compare immunogenicities of vaccine candidates. However, inclusion of convalescent patient sera as an assay control in some clinical trials makes comparisons easier and more accurate. The neutralizing antibody titers from low to high are as follows: Ad5 vectored vaccine, mRNA vaccine, and protein subunit vaccine. The cellular immunity elicited by the vaccine might play a protective role as well. Although the clinical data seem encouraging, there will be still many challenges moving forward. For example, there are few vaccine candidates in clinical trials designed to elicit IgA, which can protect the upper respiratory tract [22,86]. In fact, IgG, which can be efficiently induced by current clinical vaccine candidates and the IM administration, only protects the low respiratory tract [22]. This means that vaccine candidates may not be able to provide sterilizing immunity. Even though COVID vaccines may be developed successfully, it will be difficult to manufacture enough doses (to achieve global herd immunity, billions of vaccine doses will be needed) and the equal and effective distribution of vaccine doses will be a daunting challenge.

Author contributions

Jinghua Yan and Qingrui Huang carried out the concepts, design and definition of intellectual content. Qingrui Huang conducted literature search and manuscript preparation. Jinghua Yan carried out manuscript editing. Both authors have read and approved the content of the manuscript.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Biographies

Dr. Qingrui Huang, an associate professor in Institute of Microbiology, Chinese Academy of Sciences, China. He received Ph.D from Institute of Process Engeering, Chinese Academy of Sciences. He has been focusing on the development of vaccines against bacterium and viruses, especially mRNA vaccines and protein subunit vaccines.

Dr. Jinghua Yan, a professor and Principle Investigator in Institute of Microbiology, Chinese Academy of Sciences, China. She obtained Ph.D from Institute of Bioengineering, Academy of Military Medical Sciences. Her reserach interest mainly focuses on the development of vaccines and therapeutic antibodies against emerging viruses.