Protein-based nano-vaccines against SARS-CoV-2: Current design strategies and advances of candidate vaccines

Abstract

The outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shaken the global health system. Various nanotechnology-based strategies for vaccine development have played pivotal roles in fighting against SARS-CoV-2. Among them, the safe and effective protein-based nanoparticle (NP) platforms display a highly repetitive array of foreign antigens on their surface, which is urgent for improving the immunogenicity of vaccines. These platforms greatly improved antigen uptake by antigen presenting cells (APCs), lymph node trafficking, and B cell activation, due to the optimal size, multivalence, and versatility of NPs. In this review, we summarize the advances of protein-based NP platforms, strategies of antigen attachment, and the current progress of clinical and preclinical trials in the development of SARS-CoV-2 vaccines based on protein-based NP platforms. Importantly, the lessons learnt and design approaches developed for these NP platforms against SARS-CoV-2 also provide insights into the development of protein-based NP strategies for preventing other epidemic diseases.

Graphical abstract

1. Introduction

Since the outbreak of the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019, it has spread across more than 220 countries and regions, and resulted in more than 500 million confirmed cases (https://covid19.who.int/, accessed on 26 August 2022). As one of the most effective measures to control SARS-CoV-2 transmission. More than 12 billion vaccine doses have been administered worldwide until 23 August 2022, (https://covid19.who.int/, accessed on 23 August 2022). So far, a variety of advanced vaccine platforms have been employed for rapidly developing COVID-19 vaccine, such as mRNA, DNA, inactivated virus, live attenuated virus, viral vector (replicating and non-replicating), replicating or non-replicating viral vector combined with antigen presenting cell (APC), protein subunit, and virus-like particles (VLPs) (https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines). Based on these versatile platforms, 167 candidates of COVID-19 vaccine have entered into different phases of clinical trials (Fig. 1 ).

Fig. 1.

COVID-19 candidate vaccines based on various vaccine platforms have been developed in the current clinical trials.

Data from World Health Organization (WHO), by June 28, 2022.

Nanoparticles (NPs) with size ranging from 10 to 200 nm showed significant advantage in the uptake by antigen-presenting cells (APCs) [1], [2]. In addition, NPs provide additional beneficial properties, such as adjuvant and presentation/carrier activity, making them an ideal platform to efficiently deliver and display foreign immunogens to promote an immune response. Examples of the NP platforms with their size ranging from 10 to 30 nm for the vaccines are displayed in Fig. 2 . In general, protein-based NPs can divide into two major categories: VLPs and non-VLPs, both of which are assembled from various protein subunits in vivo or in vitro [3]. Furthermore, VLPs are classified as non-enveloped and enveloped subtype [4]. The non-enveloped VLPs are self-assembled from viral capsid proteins, and thus defined as capsid VLPs (cVLPs), whereas the enveloped VLPs (eVLPs) consists of lipid membrane derived from host cells together with viral membrane protein(s) [5].

Fig. 2.

Examples of NPs as vectors for COVID-19 vaccine development. (A) Crystal structures of capsid VLPs. 3D structures from Protein Data Bank (PDB), (MS2: 2MS2 [63], AP205: 5LQP [64] and CuMV: 1F15 [65]). (B) Crystal structures of non-VLPs derived protein self-assembling NPs. 3D structures from PDB, (Ferritin: 1MFR [66] and Lumazine Synthase: 1HQK [67]); 3D structure of I53-50 from EMDataResource (EMD-0350 [68]).

In vaccinology, VLPs may be modified by several strategies, such as genetic fusion, foreign tag coupling, or chemical conjugation, incorporation of foreign immunogens or molecular fragments onto VLPs to generate chimeric VLPs (chi-VLPs) [5]. Thus, chi-VLPs can be used as a vector to develop potential bivalent or multivalent vaccines to combat several diseases. Alternatively, non-VLP NPs may be assembled by non-virus derived native protein or human-designed protein, these proteins being capable of self-assembly into NPs with highly oligomeric structures in vitro or in vivo. This sophisticated design/platform has been employed for COVID-19 vaccine development [6], [7]. In this review, current advances in the development of SARS-CoV-2 vaccines based on protein-based NP platforms are summarized and the immune mechanisms triggered by these NP vaccines, along with distinct adjuvants in the vaccine formulae, are also reviewed.

2. Advantages of NP-based vaccines

NP-based vaccines are far more immunogenic than recombinant protein-based subunit vaccines. The NPs ranging between 10 and 200 nm in size can be efficiently taken up through various endocytic pathways, i.e. receptor-mediated endocytosis, phagocytosis and micropinocytosis, and then processed by professional antigen presenting cells (APCs), especially dendritic cells (DCs) [2]. Compared to protein subunits, which are generally neglected by DCs, the physicochemical characteristics of NPs, particularly the molecular patterns on the surface, fulfill the requirement for interactions between NPs and various receptors on the DCs. Consequently, NPs are primed for DCs detection and internalization. Then, the internalized NPs are digested into a plethora of peptides within a phagolysosome and processed through the classical antigen-processing pathway (Fig. 3A), after which the antigen peptides are presented via MHC class II or cross-presented via MHC class I on the DCs' surface [8]. Subsequently, the activated DCs migrate into the T cell zone of lymphoid tissues such as lymph nodes (LNs) or spleen to initiate T cell activation. In addition, NPs may also diffuse freely into LNs, thus, making NPs an ideal platform for delivering foreign antigens and immunostimulants. Some of the particles of 20–200 nm can efficiently enter the lymphatic system by diffusing through lymphatic endothelial cell junctions, while other particles endocytosed by DCs can enter into the lumen of the lymphatic vessels by intravasation [1].

Fig. 3.

Advantages of NP-based vaccines. (A) Adaptive immune response stimulated by NP-based vaccines. (B) NP-based vaccines stimulated robust B cell activation via the interaction of multiple antigens with BCRs.

Highly specialized uptake and processing of NPs by APCs, particularly the DCs, are crucial for the induction of T cell-mediated immune response. The specific and direct binding of a peptide-MHC-II complex to the T cell receptor (TCR) is a prerequisite for T cell activation, and the binding strength (affinity) plays a critical role in T cell activation. Nonetheless, some NPs may traffic rapidly into neighboring LNs via the afferent lymphatic vessels as fast-draining particles due to their nanometer property. Apparently, 20 nm particles can transit rapidly to the B cell zone of LNs in 2 h, while particles of 500 nm size will take 24 h to reach the LN [9]. Once NPs penetrate into the B cell zone, the molecular pattern of the repetitive array of antigens on the NP surface bind and then trigger the cross-linking of the B cell receptors (BCRs) (Fig. 3B), leading to a robust B cell activation [10]. A series of events occur upon the B cell activation. These include B cell proliferation and migration, upregulation of MHC-II molecules, activation of the cognate T_H cells, production and secretion of IgM and IgG and generation of specific long-lived memory B cells [11]. Furthermore, the processed viral peptides can also efficiently load on MHC-I via cross-presentation and present to CD8⁺ T cells by DCs. This can trigger cytotoxic T lymphocyte (CTL) mediated cellular responses to eliminate intracellular pathogens [12]. Of note, NPs can intrinsically act as adjuvants by utilizing their nano-size and highly repetitive antigen structures, which can enhance their binding to the low-affinity BCRs through multivalent interactions (Fig. 3B). Thus, NPs even without adjuvants are capable of inducing durable B cell responses and high-level of antibody titers, and directly activate B cells at much lower concentrations than antigens in the form of monomeric proteins [13].

3. Strategies of foreign antigen attachment to various NPs

Currently, at least 3 different strategies were employed to display various foreign antigens on the NP: genetic fusion, tag coupling, and chemical conjugation (Fig. 4 ). These strategies allow NP platforms to be decorated with various foreign antigens, resulting in the increase of antigen presentation and particle size. Genetic fusion and tag coupling display foreign antigens specifically to the N- or C-termini of the protein subunits using flexible connection linkers (such as a GS linker) and present a more precise antigen arrangement (Fig. 4A). Generally, genetic fusion is the most convenient method to display foreign antigens since this method can simply target two termini of each subunit of the NPs. However, some challenges still existed as fused antigens may adversely affect expression level and the proper folding of recombinant proteins even compromise NPs assembly. In contrast, tag coupling presentation strategy allows independent protein expression and modular attachment of foreign antigens onto the surface of the NPs. It may be accomplished by combining a binding tag to a terminus of a foreign antigen and a catcher or receptor fused to the surface of NPs (Fig. 4B). The most popular tag coupling systems is SpyTag/SpyCatcher. The SpyTag/SpyCatcher system containing SpyTag (13 aa) and SpyCatcher (113 aa) has been extensively used for foreign antigen presentation. SpyCatcher fused to either the N- or C-terminal end of the NP subunit can form an irreversible bond with SpyTag fused to foreign antigens or vice versa. Overall, tag coupling strategy allows rapid modular attachment of various foreign proteins to the NP platform, compared to the genetic fusion method. In contrast, chemical conjugation depends on chemically crosslinking foreign antigens to the NPs, usually leading to an uneven antigen arrangement (Fig. 4C). In this method, the foreign antigen containing a free cysteine residue (or via engineering) is crosslinked to the surface-exposed lysines of the NP using an amine- and sulfhydryl-reactive bifunctional cross-linker, such as succinimidyl 6-β-maleimidopropionamido hexanoate (SMPH). Compared with other methods, the uneven decoration of chemical conjugation may result in poor display of the foreign antigens on the NPs surface and consequently inefficient B cell activation. Nevertheless, the chemical conjugation has been used as an efficient method to attach foreign protein-based antigens to significantly enhance immune responses.

Fig. 4.

Attachment strategies of foreign antigen to NP platforms.

4. COVID-19 VLP vaccines in clinical trials

The structural proteins of SARS-CoV-2 include 4 viral proteins of spike (S), membrane (M), nucleocapsid (N), and envelope (E) proteins. The S protein functions as homotrimers on the viral membrane to mediate viral entry into host cells. This protein contains an ectodomain, a transmembrane domain (TM), and a short cytoplasmic tail region (CT). The ectodomain is composed of two subunits (S1 and S2). The receptor binding domain (RBD) located in the carboxyl-terminus of the S1 subunit, can directly bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell membranes. The full-length S protein and its receptor binding domain (RBD) are both capable of eliciting robust neutralizing antibodies (NAbs) against SARS-CoV-2 infection. Thus, both of them have been widely chosen as the protective antigen for COVID-19 vaccine designs. For instance, both mRNA and adenovirus-based vaccines endogenously express the S protein in host cells, eliciting the protective immune responses. Alternatively, in vitro purified S protein or the RBD was cross-linked onto the surface of various NPs to generate multivalent NP-based vaccines, such as VLPs (Fig. 5 ).

Fig. 5.

Protein-based NP vaccines for COVID-19. (A) Self-assembled VLPs expressed full-length S protein alone, co-expression S, M and E or S, M, E, and N proteins. (B) Chi-eVLPs chimeric S ectodomain by TM/CT grafting incorporated into other viral enveloped proteins. (C) Chi-cVLPs or non-VLP NPs as vector to display RBD, RBM or S ectodomain.

To date, 6 VLP-based COVID-19 vaccines in clinical trials are listed by the World Health Organization (WHO), including 2 self-assembled VLPs, murine leukemia virus (MLV)-based chimeric eVLPs (the ectodomain of the S protein), and 3 cVLPs displaying SARS-CoV-2 RBD vaccines (Table 1 ). Coronavirus-like particle (CoVLP) containing S protein designed by Medicago Inc. was generated in a plant system of Nicotiana benthamiana. The stabilized prefusion S protein with point mutations is capable of spontaneously assembling into CoVLP after purification in vitro. Of note, the spherical CoVLP with a diameter of 80–120 nm is similar to the native viral structure [14]. In addition, CoVLP vaccine is formulated with two types of adjuvants: (1) AS03 adjuvant system (GlaxoSmithKline, GSK) and (2) CpG 1018 (Dynavax), respectively. CpG 1018 is a well defined Toll-like receptor 9 agonist that favors strong Th1 immune response [15]. In a phase 1 clinical trial (NCT04450004), 3 different doses (3.75, 7.5 and 15 μg) of CoVLP unadjuvanted or adjuvanted with either AS03 or CpG 1018 were administrated in adults. The study indicated that dosage-independent NAb titers induced by adjuvanted CoVLP vaccines were significantly higher than that of the unadjuvanted vaccines. Furthermore, the NAb titer induced by CoVLP adjuvanted with AS03 after the second dose was 10-fold higher than the titer of COVID-19 convalescent sera. The CoVLP vaccine also induced strong S protein-specific cell-mediated immune responses [14]. With these encouraging results in the phase 1 and phase 2/3 clinical trials, the CoVLP vaccine entered a phase 3 study (NCT05040789), in which two intramuscular injections with dose of CoVLP (3.75 μg) adjuvanted with AS03 were administered. The clinical trial was scheduled to be completed by May 31, 2022.

Table 1.

VLP-based candidate vaccines for COVID-19 in clinical trials.

Antigen	Production system	Adjuvant	Developer	Phase	Clinical trial registration
Full-length S protein self-assembling Coronavirus-like particle (CoVLP)	Nicotiana benthamiana	CpG 1018/AS03	Medicago Inc.	Phase 3	NCT04450004 (Phase 1) NCT04662697 (Phase 2) NCT04636697 (Phase 2/3) NCT05040789 (Phase 3)
S, M, E and N protein self-assembling VLPs	HEK-293T cells	CpGODN-K3/Alum	The Scientific and Technological Research Council of Turkey	Phase 2	NCT04818281 (Phase 1) NCT04962893 (Phase 2)
Murine leukemia virus (MLV)-based eVLPs chimeric spike ectodomain (VBI-2902a)	HEK-293SF-3F6 GMP compliant cells	Aluminum phosphate adjuvant	VBI Vaccines Inc.	Phase ½	NCT04773665
HBsAg VLP display RBD	Yeast	Alum + CpG 1018/Alum alone	Serum Institute of India + Accelagen Pty + SpyBiotech	Phase ½	ACTRN12620000817943 ACTRN12620001308987
AdaptVac's capsid VLP technology (AP205) display RBD (ABNCoV2)	Drosophila S2 insect cells (ExpreS2)	With or without adjuvant MF59	Radboud University	Phase 1	NCT04839146 (Phase 1)
RBD	/	Aluminum hydroxide	Yantai Patronus Biotech Co., Ltd.	Phase 1	NCT05125926 (Phase 1)

COVID-19 VLPs vaccines were collected from WHO COVID-19 vaccine tracker and landscape (COVID-19 vaccine tracker and landscape (who.int), accessed on Jun 28, 2022). Note: “/” no information.

SARS-CoV2 VLPs have also been generated by transiently co-transfecting 4 viral genes encoding structural proteins (S, M, E and N) into HEK-293 cells. The size of VLPs is similar to the authentic virus. Compared with sole alum or CpGOND-K3 adjuvanted vaccine, after adsorbing on the alum, the CpGOND-K3-adjuvanted VLPs elicited both robust NAbs and Th1-biased T cell responses, showing a successful protective immunity against the challenge of SARS-CoV-2 in K18 hACE2 Tg mice with reduced virus load and less lung lesions [16]. In phase 1 clinical trial (NCT04818281), 2 different doses (10 and 40 μg) of alum adsorbed CpGOND-K3-adjuvanted VLP vaccine was administrated to 12 participants of each group (https://clinicaltrials.gov/ct2/show/NCT04818281). In the phase 2 trial (NCT04962893), the immunogenicity of three VLP vaccines based on authentic SARS-CoV-2 or Alpha variant and their combination was administered to 110 participants of each group in two doses with a 21-day interval on July 15, 2021 (https://clinicaltrials.gov/ct2/show/NCT04962893).

Of the 6 COVID-19 VLP vaccines in clinical trials, two murine leukemia virus (MLV)-based chimeric eVLPs (the ectodomain of the S protein), named VBI-2902a (Wuhan strain) and VBI-2905a (B.1.351 variant), respectively, are in phase 1/2 clinical trials, currently (https://clinicaltrials.gov/ct2/show/NCT04773665). In a preclinical trial, VBI-2902a vaccine adjuvanted with aluminum phosphate elicited high NAb titers with balanced IgG1 and IgG2a antibody responses for more than 3 months after a single-dose immunization, which protected Syrian golden hamsters from challenge of SARS-CoV-2 [17]. The result suggested that VBI-2902a vaccine has a great potential to stimulate immune responses against SARS-CoV-2 and warrants on-going clinical evaluation.

In addition, the RBD derived from the truncated S protein of SARS-CoV-2 has been extensively used in the COVID-19 vaccine development. However, the antigenicity of the RBD is poor in the absence of adjuvants. In order to elicit a stronger immune response, RBD was displayed on the surface of cVLPs to generate chimeric cVLPs (chi-cVLPs). Hepatitis B surface antigen (HBsAg) VLPs, an ideal cVLP vector, which exhibit good safety and immunogenicity. Two human hepatitis B virus (HBV) commercial vaccines based on HBsAg VLPs have been successfully developed by Merck (Recombivax HB®) [18] and GSK (Engerix®-B) [19], and the first licensed malaria vaccine (Mosquirix®) developed by GSK also used HBsAg VLPs fused with RTS, S proteins derived from Plasmodium falciparum [20]. Thus, the RBD SARS-CoV-2/HBsAg VLP vaccine was designed to prepare chi-cVLP vaccine. The HBsAg VLPs platform was used to display the RBD antigen and efficiently induce RBD-specific antibodies. In a phase 1/2 randomized clinical trial (ACTRN12620001308987), the VLPs adjuvanted with Alum + CpG 1018 or Alum alone was administered as two doses at an interval of 28 days to evaluate the safety and immunogenicity in healthy adults (https://anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12620001308987).

The phage derived AP205 VLPs is a promising scaffold for displaying the SARS-CoV-2 RBD. Using AdaptVac's capsid VLP technology (Tag/Catcher), two RBD antigens (genetically fused at the N- or C-terminus of AP205 capsid protein, defined as RBDn-CLP and RBDc-CLP, respectively) were displayed on the AP205 VLPs surface for inducing NAb titers [21]. The RBDn-CLP demonstrated high stability and immunogenicity in the mice compared with RBDc-CLP. Thus, the RBDn-CLP vaccine (named ABNCoV2) was selected as a candidate for clinical trial study. In the phase 1 trial (NCT04839146), ABNCoV2 vaccine was administered with or without MF59 adjuvant in healthy adults on March 11, 2021, and 5 doses (6 μg, 12 μg, 25 μg, 50 μg and 70 μg) of the cVLPs were used to evaluate its safety and tolerability (https://clinicaltrials.gov/ct2/show/NCT04839146). The clinical evaluation is on-going and the immunogenicity results are yet to be published.

The SARS-CoV-2 vaccine (LYB001), developed by Yantai Patronus Biotech Co., Ltd., consists of RBD from SARS-CoV-2 and VLP vector, adjuvanted with aluminum hydroxide (https://clinicaltrials.gov/ct2/show/record/NCT05125926). This vaccine is currently in a phase 1 clinical trial (NCT05125926) to evaluate its safety and immunogenicity.

5. Advance of NP-based COVID-19 vaccines

5.1. Self-assembled COVID-19 VLPs

Although traditional vaccines based on live-attenuated or inactivated pathogens have been widely employed clinically, concerns about their biosafety and efficacy remain central topics of vaccine development. In addition, the components of these vaccines are not fully defined and their potential side effects and contribution to immunoprotections are unknown. VLPs formed by self-assembling viral structural proteins (capsid or envelope proteins), ranging from 20 to 200 nm in diameter, are safe vaccine candidates due to the absence viral genomes. Importantly, these VLPs can induce robust innate and adaptive immune responses in hosts due to the similar structural and antigenic properties with authentic viruses. Therefore, VLP-based nanoscale particles are promising vaccine candidates.

Recently, SARS-CoV-2 VLPs have been prepared from both mammalian cell and insect cell systems, and the immunogenicity of the VLPs have been evaluated in animal models. The strategic design of the SARS-CoV-2 VLPs and the immune responses to these VLPs in animal models are shown in detail in Table 2 . Apparently, SARS-CoV-2 VLPs can be generated by co-expression of 4 of proteins (S, M, E and N) [16], [22] or 3 of proteins (S, M and E) [23], [24], [25]. For instance, RQ3013-VLP is formulated from a cocktail of mRNAs encoding S, M and E proteins that can induce both NAbs and robust VLPs-specific T cell responses in BALB/c mice, by using lipid nanoparticles (LNPs) package system [25]. In addition, VLP-based on the full-length S protein have been reported [26]. Furthermore, the S protein alone is capable of inducing the formation of the VLPs and elicited high-level of S protein-specific IgG and Th1-biased IgG2a antibody response without adjuvant in mice [26].

Table 2.

List of VLP or NP-based vaccines against SARS-CoV-2.

Platform	Scaffold	Antigen (s)	Production system	Linking strategy	Animal model	Adjuvant	Reference
VLPs	None	S, M, E and N	Vero E6 & HEK-293 T cells	None	/	/	[22]
	None	S, M and E	293 T cells	None	/	/	[23]
	None	S, M and E	Baculovirus-ExpiSf9™ cells	None	/	/	[24]
	None	S, M and E	HEK-293A cells	None	BALB/c mice	LNPs package system	[25]
	None	S	Baculovirus-Sf9 cells	None	Mice	Without adjuvant	[26]
Chi-eVLPs	Influenza A virus M1	S1 subunit	Baculovirus-Sf9 cells	None	Mice	Without adjuvant	[26]
	NDV	S ectodomain	HEK-293 T cells	None	BALB/c mice	SAS	[30]
Chi-cVLPs	AP205	RBM	E. coli	Genetic fusion	BALB/c mice	Without adjuvant	[31]
	MS2	S ectodomain	Expi293F cells	Biotin-streptavidin	Hamsters	Alhydrogel	[32]
	CuMVTT	RBM	E. coli	Genetic fusion	BALB/c mice	Without adjuvant	[36]
	CuMVTT	RBD	Expi293 F cells	Chemically coupling	BALB/c mice	Without adjuvant	[37]
NP	LS	RBD	293F cells	Fc-tagged coupling	BALB/c mice	Aluminum hydroxide, MPLA	[40]
	LS	S ectodomain	Expi293F cells	SpyTag/SpyCatcher	BALB/c mice	SAS	[41]
	Ferritin	RBD	293i cells	Genetic fusion	Rhesus monkeys	Aluminum	[42]
	Ferritin	RBD	HEK-293T cells	Genetic fusion	Ferrets	AddaVax	[43]
	Ferritin	S ectodomain	Expi293F cells	Genetic fusion	BALB/c mice	Quil-A, MPLA	[44]
	Ferritin	RBD	HEK-293T cells	Genetic fusion	C57BL/6 mice	LNPs package system	[45]
	Ferritin	RBD	293F cells	SpyTag/SpyCatcher	C57BL/6 mice	CpG-1826	[46]
	Ferritin	RBD, RBD-HR	CHO-S cells	SpyTag/SpyCatcher	Mice, rhesus macaques	SAS	[47]
	Ferritin	RBD, S ectodomain	Expi293 cells	SpyTag/SpyCatcher	K18-ACE2 Tg mice	CpG	[48]
	Ferritin	RBD	HEK-293 F cells	SpyTag/SpyCatcher	BALB/c mice	AddaVax, SAS	[54]
	mi3	RBD	Expi293F cells	SpyTag/SpyCatcher	BALB/c, C57BL/6 mice, pigs	AddaVax	[52]
	mi3	RBD	Expi293F cells	SpyTag/SpyCatcher	Mice	AddaVax	[53]
	mi3	Multivalent RBD	Expi293F cells	SpyTag/SpyCatcher	Mice	AddaVax	[60]
	mi3	RBD	HEK-293 F cells	SpyTag/SpyCatcher	BALB/c mice	AddaVax, SAS	[54]
	mi3	HR2-deleted S	ExpiCHO cells	SpyTag/SpyCatcher	BALB/c mice	AddaVax, Adju-Phos	[55]
	I53–50	RBD	HEK-293 F cells	SpyTag/SpyCatcher	BALB/c mice	AddaVax, SAS	[54]
	I53–50	S ectodomain	HEK-293 F cells	Genetic fusion	BALB/c mice, rabbits, cynomolgus macaques	Poly-IC, squalene emulsion adjuvant, MPLA liposomes	[56]
	I53–50	RBD	Expi293F cells	Genetic fusion	BALB/c mice, pigtail macaques	AddaVax	[57]
	I53–50	Multivalent RBD	Expi293F cells	Genetic fusion	BALB/c mice, pigtail/ rhesus macaques	AddaVax, AS03	[61]
	I53–50	Multivalent S	Expi293F cells	Genetic fusion	BALB/c mice, cynomolgus macaques	MF59	[62]

NDV: Newcastle disease virus; CuMV: cucumber mosaic virus; HR: heptad repeat; LS: lumazine synthase; LNPs: lipid nanoparticles; SAS: Sigma Adjuvant System; MPLA: monophosphoryl lipid A; RBD: receptor-binding domain; RBM: receptor binding motif; Note: “/” represented not tested.

5.2. Chimeric enveloped VLPs

Many outer membrane proteins derived from enveloped viruses spontaneously can form VLPs (eVLPs) in cell cultures or in the host. For example, eVLP formed by the surface protein of HBV have been successfully developed into a commercial vaccine against HBV infection. Furthermore, the extracellular domain of the surface protein can be replaced by foreign proteins or epitopes from other pathogens without compromising VLP formation. We refer the VLPs as chimeric eVLPs (chi-eVLPs), typically for those with foreign antigens/epitopes display on the surface for binding to BCR. A SARS-CoV-2 VLPs, designated as CoVLP, was prepared by expression of the SARS-CoV-2 S protein in N. benthamiana using A. tumifaciens transfection by Medicago LLC. This VLPs is a typical chi-eVLPs since both TM/CT and signal peptide of the S protein were replaced with the TM/CT derived from the haemagglutinin (HA) of H5N1 influenza and the signal peptide of the protein disulfide isomerase of alfalfa, respectively, to increase the assembly and budding of the chi-eVLPs.

Influenza Matrix 1 (M1) self-assembled VLPs (120 nm in diameter) is another common eVLP platform, which has been successfully used for influenza vaccines development. M1 interacting with the CT region of hemagglutinin (HA) and neuraminidase (NA) proteins is critical to facilitate assembly and formation of VLPs [27]. Similarly, M1-based eVLPs consisting of M1 and the chimeric S protein ectodomain have been used in SARS-CoV and MERS-CoV vaccine development [28], [29]. Recently, VLPs were formed by co-expression of the SARS-CoV-2 S1 subunit fused with HA TM/CT regions of influenza A virus and M1. The Chi-eVLPs elicited high levels of S protein-specific IgG in a mouse model (Table 2) [26]. Replacement of the S protein TM/CT regions with the TM/CT derived from HA or NA allowed the antigen anchoring into the lipid membrane. Therefore, M1-based eVLPs might be employed for presentations of various foreign surface glycoproteins or envelope proteins of other pathogens. Moreover, this design strategy can be adopted for developing multivalent vaccines to simultaneously combat several infectious diseases. Recently, a novel chi-eVLPs generated by a chimeric protein expression, in which the ectodomain of the SARS-CoV-2 S protein (prefusion-stabilized via two proline substitutions at residues 986–987: S2P) was fused with the transmembrane and cytoplasmic domains of the Newcastle disease virus fusion protein (F), elicited substantial immune response in mice [30].

5.3. Chimeric capsid VLPs

cVLPs, assembled by multiple capsid protein (Cap) monomers are promising NP scaffolds for presenting various foreign antigens. Genetic fusion of foreign antigens to NP is a direct method. This method can provide great precision via site-specific antigen attachment, which is accessible and properly orients the antigens. Presented antigen copies is consistent with Cap monomers. Thus, foreign antigens were genetically fused on the chi-cVLPs surface, thereby decided the potential of inducing humoral immune response.

Bacteriophages are viruses that only infect bacteria, but not human. Therefore, bacteriophage-derived VLP platforms (i.e. AP205 and MS2) are promise to offer potential scaffolds for COVID-19 vaccine development. Both AP205 and MS2 contain 180 monomers of the coat proteins, which are able to oligomerize and form an icosahedral capsid structure. Attachment of SARS-CoV-2 RBM to the Cap C-terminus of AP205 cVLPs without adjuvant induced NAbs in BALB/c mice [31]. In another study, biotinylated prefusion-stabilized S protein ectodomain was attached to MS2 VLPs coated with streptavidin [32]. This Chi-cVLPs formulated with alhydrogel adjuvant elicited significantly higher NAb titers after a single immunization and protected hamsters from SARS-CoV-2 virus challenge.

Cucumber mosaic virus (CuMV), a plant virus with a diameter of 30 nm in size, contains self-assembling icosahedral capsid. CuMV VLPs capable of displaying foreign protective antigens have been studied in the development of hepatitis C virus (HCV), Zika virus, and malaria vaccines [33], [34]. A newly chimeric CuMV_TT-VLPs, into which a universal tetanus toxin (TT) T_H cell epitope was inserted, enhanced the interaction between T_H cells and B cells and is expected to significantly improve immune responses [35]. The CuMV_TT-VLPs displaying the SARS-CoV-2 S RBM on its surface has elicited both RBD-specific IgG and IgA antibodies as well as a strong viral NAb response in BALB/c mice [36]. In another case, a highly repetitive display of the RBD on CuMV_TT VLPs by chemical coupling has also elicited high levels of NAbs in mice as a lead candidate vaccine [37].

5.4. Non-VLPs derived protein self-assembling NP platforms in COVID-19 vaccines

Besides VLP-derived NP platforms, non-viral derived protein self-assembling NPs have been extensively employed as the new platforms for the development of COVID-19 vaccines (Table 2). These platforms are commonly based on enzymes or proteins that readily self-assemble into stable oligomeric structure.

Lumazine synthase (LS) is an enzyme involved in riboflavin synthesis, and 60 of the monomers are capable of self-assembling into an NP with a diameter of 15 nm in size. LS has been developed as a scaffold for NP-based vaccines and offer the promise to enhance the efficacy of presenting foreign antigens in vivo [38], [39]. Both N- and C-termini are structurally surface-exposed allowing the presentation of 60 copies of foreign immunogens. An Fc-tagged dimeric RBD of the SARS-CoV-2 S bound to protein A tagged LS can form 120 copies of the RBD on the LS-based NP's surface. This significantly elicited strong and long-lasting (more than 2 months) NAbs can protect mice against high-titer SARS-CoV-2 infection [40]. In addition, LS has also been successfully used to display SARS-CoV-2 spike trimer on its surface by using the Expi293F cell secretion system, which significantly enhanced B cell stimulated response and elicited higher NAb titers than spike protein alone in mice [41].

Ferritin, widely expressed in living organisms, can self-assemble into a spherical NP (~12 nm in diameter) containing 24 of the subunits. This non-VLP NPs with small size is a well-established and robust vaccine platform/scaffold displaying various foreign antigens against distinct pathogens, including HBV, HCV, Enterovirus 71 virus, and MERS-CoV etc. Particularly, this NP can induce strong humoral immune responses against these foreign antigens [2]. Recent studies demonstrated that presentation of RBD, trimeric RBD or the whole ectodomain of the SARS-CoV-2 S on the ferritin-based NP significantly improved both humoral and T cell-mediated immune responses and provided an effective protection against SARS-CoV-2 challenge in animal models [42], [43], [44], [45]. Furthermore, the SpyTag/SpyCatcher system has also been successfully used for presenting SARS-CoV-2 RBD, RBD-HR or spike protein on the ferritin NP surface [46], [47], [48].

The engineered i3-01 NP derived from thermophilic bacteria can spontaneously self-assemble into a dodecahedral particle (25 nm in diameter and 60 subunits) [49]. Recently, a mutated i3-01 (mi3) was computationally designed to improve particle uniformity and stability, thereby enhancing the NP's immunogenicity [50]. This kind of NPs have been genetically fused with SARS-CoV-2 RBD for novel vaccine development [50], [51]. The SARS-CoV-2 RBD presented on mi3 NP surface induced much stronger NAb response than did RBD alone [52], [53], [54]. In addition, an optimized S antigen (HR2-deleted glycine-capped spike, S2GΔHR2) was multivalent displayed on NPs (i3-01 and E2p) using the SpyTag/SpyCatcher system, and these multilayered S2GΔHR2 i3-01 and E2p NPs elicited up to 10-fold higher NAb titers and induced a strong T cell response benefit for further protective cellular immunity [55]. Importantly, S2GΔHR2 with higher metastability than S2P and provided an effective protein vaccine candidates. Similarly, two-component, I53–50, presented more antigen copies (120) compared to mi3 (60). I53-50 has also been developed as a COVID-19 candidate vaccine in Neil P. King's group. They genetically engineered the stabilized prefusion S protein to the I53–50 NP surface, and the NP induced a potent NAb response and protected macaques against a high-dose SARS-CoV-2 challenge [56]. Of note, presentation of RBD antigen on I53-50 induced 10-fold higher NAb titers than the prefusion-stabilized spike alone, despite using a 5-fold lower dose [57]. These results suggest that NP-based vaccines may engage BCRs via multiple cross-linking and strongly activate B cells at even much lower concentrations than the concomitant monomeric antigens.

Numerous zoonotic coronaviruses, such as SARS-CoV and MERS-CoV, have emerged in the past 20 years. Likewise, SARS-like coronaviruses circulating in bats (bat WIV1 and SHC014 strains) are thought to represent an ongoing threat to humans [58], [59]. More efforts are needed to develop a universal vaccine to control seasonal and new coronavirus pandemic outbreaks. To design a universal vaccine against a broad spectrum of coronaviruses, Pamela J. Bjorkman's group developed mosaic mi3 NPs displaying the RBD of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from zoonotic coronaviruses [60]. This mosaic NP vaccine induced NAbs with superior cross-recognition of heterologous RBDs simultaneously protecting against SARS-CoV-2 and other emerging zoonotic coronaviruses. Similarly, another group designed mosaic and cocktail RBD NP vaccines that display multiple sarbecovirus RBDs to induce broader immune responses against heterologous sarbecovirus challenge, paving the way for a next generation of universal sarbecovirus vaccines [61]. Moreover, displaying S from SARS-CoV-2 prototype and three major variants on the NP surface, forming a quadrivalent mosaic SARS-CoV-2 NP vaccine, elicited a breadth of protective humoral immune responses against multiple SARS-CoV-2 variants [62]. These results suggest that the versatility of NP platforms for virus antigen presentation during vaccine development could be an ideal approach to the development of vaccines against a possible future pandemic of emergent zoonotic coronaviruses.

6. Conclusion

Currently, the continuous development of a new generation of precise vaccines to combat the pandemic of COVID-19 is a compelling task of scientists. Although inactivated vaccine and mRNA are effective, protein-based NP vaccines remain the safest and effective design approach. In general, protein subunit vaccines will require adjuvants to be efficacious. However, protein-based NPs with optimal size and the high density (multivalence) of antigens presented on the surface of NPs can elicit higher NAb titers than monomer proteins. According to the above reviewed data, we believe that protein-based NPs can serve as effective platforms for the production of novel vaccines that range from classical to next-generation vaccines.

As of the writing of this review, six kinds of COVID-19 NP-based candidate vaccines are in various stages of clinical trials, with several NP vaccines that are in preclinical study, including self-assembled VLPs, chi-eVLPs, and protective RBD or spike antigens attached to NP, which are also being developed. However, several factors are important for COVID-19 NP-based vaccine design and development. Generally, it is extremely critical to display antigens with correct conformation on NPs surface to elicit antigen-specific high affinity NAbs, while the stability of engineered NPs can determine the level of antigenicity. Thus, NPs can be engineered with critical mutations to modify its physicochemical properties to improve thermal stability without compromising its oligomeric structure. In addition, as a foreign vector, safety is the key factor limiting the leap from preclinical development to clinical trials. Despite these challenges, NP-based vaccines represent one of the most promising approaches to next-generation vaccine development. Particularly, as several new variants of SARS-CoV-2 are emerging worldwide, and currently vaccines provide less protections against them. NPs may be further modified by genetic engineering strategies and protective immunogens derived from variants may be incorporated into NPs to generate bivalent or multivalent vaccines against these variants, simultaneously. Vaccination with more than one dose may provide satisfactory protection, eliminating the side effects from multi-immunizations. Thus, people can be vaccinated by a combination of different antigens to protect against multiple variants. In conclusion, human health is challenged by the continuous emergence of SARS-CoV-2 new variants, and an effective vaccine is urgently needed. Given the size, multivalence and versatility of NPs, we expect they will play vital roles in the development of next-generation vaccines against the pandemic of COVID-19 or other infectious diseases.

CRediT authorship contribution statement

DW and Yi. Y outlined the manuscript and designed the tables and figs. DW, Yo. Y, BL, NE, and Yi. Y contributed to writing the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by National Natural Science Foundation of China (No. 32172844) and Hunan Province Technology Breakthrough Project of 2021 for the open competition mechanism to select the best candidates (No. 2021NK1030).

Declaration of competing interest

No conflict of interest declared.

Data availability

No data was used for the research described in the article.