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Protein Nanoparticles as Viral Vaccine Platforms

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Submitted:

07 May 2024

Posted:

10 May 2024

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Abstract
Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprising of killed, inactivated, or live attenuated pathogens have resulted in protective immune responses, negative consequences of their administration have been well-appreciated. Modern vaccines have evolved to contain purified antigenic proteins, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to modern vaccine platforms. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterization of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious diseases and enhancing vaccine efficacy and safety.
Keywords: 
Subject: Biology and Life Sciences  -   Virology

1. Introduction

The early report of vaccination dates to over 250 years when Thomas Dimsdale introduced powdered scabby pustules containing variola virus into the arm of Catherine the Great, the empress of Russia to protect her from smallpox. However, the British physician Edward Jenner was credited in 1796 for bringing the vaccination to mainstream medical practice using cowpox inoculation for against smallpox. Since then, vaccination has saved millions of lives affected by pathogens including viruses such as smallpox, polio, influenza, rabies, measles, just to name a few and most recently, the SARS-CoV-2. Among the variety of vaccines that have been developed and used are the conventional vaccines based on live-attenuated or killed viruses, and the subunit vaccines containing one or more of the viral antigens. Considerations for their development include attenuation of their pathogenicity, complete pathogen inactivation for safety, and preservation of potential epitopes responsible for generating effective immunity [1,2]. In recent times, conventional vaccines have faced significant shortcomings, including insufficient immune stimulation, in vivo instability, potential toxicity, and the requirement for multiple doses [2]. To overcome some of these challenges, nanotechnology platforms offer innovative solutions to enhance safety and vaccine efficacy. The engineering of nanoparticles (NPs) as delivery systems for antigens can improve antigen stability, target specificity for immune cells, control antigen release kinetics, and can be a potential gamechanger in vaccinology [3].
Several types of NPs have been employed in the field of vaccinology and targeted delivery of therapeutic agents. The inorganic NPs (INPs) can be designed to various shapes and sizes for optimal delivery and/or immunological responses. Although in some cases they exhibit adjuvant properties, they are often not biodegradable [4] and may exhibit toxicity. The organic NPs (ONPs) are derived from biomacromolecules such as lipids, producing lipid NPs (LNPs) that can encapsulate nucleic acids encoding antigens, proteins, therapeutic drugs, etc.; proteins, generating protein NPs (PNPs) that can encapsulate and/or display antigenic proteins for immunological responses; and carbohydrates, with polymeric matrices such as poly-lactic-co-glycolic acid (PLGA) forming NPs. Due to their biocompatibility, biodegradability, and low toxicity, the PNPs have been preferred over other NPs for a variety of applications including in vivo and in vitro molecular imaging, gene therapy, biosensing, targeted and non-targeted drug delivery, vaccine platforms, photodynamic and photothermal therapy, MRI contrast agent [5].
Due to the availability of genomic sequence data for many organisms of prokaryotic and eukaryotic origin and powerful tools for genomic data mining, it has become relatively easier to identify and characterize naturally occurring PNPs with novel functions. For example, phylogenetic and bioinformatics analyses coupled with biochemical studies have resulted in the identification of over 900 potential encapsulin nanocompartments in prokaryotes, some of which play critical roles in iron mineralization, oxidative and nitrosative stress resistance, and anaerobic ammonium oxidation [6]. However, it should be noted that although the existence of many PNPs in various domains of life are predicted, only a few of them have been characterized structurally and functionally in greater detail. Some of these well-characterized PNPs are being used in various applications including imaging, diagnostics, targeted drug delivery, as well as vaccine platforms [5,7,8,9,10]. The use of naturally occurring proteins that form NPs and computationally designed synthetic proteins forming NPs are emerging as new players in vaccine technology. With the global health landscape continuously impacted by emerging and reemerging pathogens causing epidemics and pandemics, nanotechnology-based vaccines will provide a rapid and adaptable platform to quickly respond to novel pathogen threats.
Here, we review the status of protein-based nanotechnology approach in revolutionizing vaccine development. Specifically, we emphasize the potential of naturally occurring proteins such as ferritin, lumazine synthase, and encapsulin as well as other nanoparticles including those derived from the capsid proteins of viruses, bacteriophages, and computationally designed synthetic protein-based NPs as emerging vaccine platforms. These proteins serve as promising building blocks for the design and construction of advanced vaccine candidates. We highlight their suitability as carriers for antigens and as adjuvants through examination of their characteristics, and immunogenic properties. Furthermore, we discuss the innovative methodologies employed to engineer these PNPs and explore their applications in enhancing vaccine efficacy, immunogenicity, and safety. As the field of nanotechnology continues to evolve, this review underscores the significant impact and potential future directions for utilizing protein-based NPs to address the pressing challenges of vaccine development.

2. Advantages of PNP-Based Vaccine Platforms and Potential Limitations

Most PNPs are composed of many copies of self-assembling protein subunits from natural sources and are particulate in nature. The precise assembly of the individual protein subunits can result in a variety of well-characterized protein architectures such as fibers, rings, tubes, catenanes, knots, and cages [11], among which the protein cages or NPs have been widely used as vaccine platforms.
Compared to the soluble monomeric antigens, the PNP-based particulate antigens offer significant advantages including the following. (i) They are of uniform shape and size; their assembly from individual subunits is highly reproducible, and they can be readily purified to near homogeneity using standardized protocols. (ii) The PNPs can be easily functionalized through genetic or chemical modifications at desired locations. As the structures of many PNPs and their subunits have been solved at atomic resolution, they can be modified accordingly to generate stable structures with presentation of antigens for optimal immune response. Modifications can be introduced at the surface, the interior, or at the subunit interface [12], which confer significant advantages for use of PNPs as vaccine platforms. Due to modularity and precise locations at which the PNPs can be functionalized, they offer enormous advantages as platform for presentation of antigens to the immune system. (iii) They are biocompatible, exhibit low toxicity, and readily biodegradable. (iv) Like viruses and virus-like particles (VLPs), the PNPs can be taken up by cells through endocytic process, an important attribute for a vaccine platform. Unlike the soluble antigens, PNPs displaying antigens exhibit enhanced trafficking in lymph nodes, are efficiently internalized by antigen presenting cells (APCs), and are known to persist longer, which are also important factors for stronger humoral and cellular immune responses [13,14,15,16]. (v) Since our immune system responds efficiently to immunogens with sizes in the nanometer range [17,18], responses to PNPs displaying multiple copies of an antigen are expected to be far higher than those of a soluble antigen. (vi) As repetitive array of an antigen improves immunogenicity and leads to enhanced immunological responses [19,20,21], the PNPs are desirable platforms for generating efficacious vaccines. PNP platforms deliver an ordered array of the antigen that can result in stronger interactions with multiple B-cell receptors (BCRs), which is critical for downstream signaling for potent B cell activation as well as antibody maturation [13,20,22,23]. (vii) PNPs smaller than 100 nm are readily taken up by peripheral or lymph node dendritic cells [24] for presentation of the antigen to trigger T cell immune responses [25,26]. (viii) Many PNP-based platforms display adjuvant properties [27,28,29,30,31], so vaccine delivery based on PNP platform allows co-administration of the antigen as well as the adjuvant simultaneously to the same immune cell for more robust immunological responses. (ix) Unlike the soluble antigens that can readily diffuse through the membranes of the blood endothelium and enter the blood circulation for faster systemic distribution, the particulate PNP-based vaccines have limited systemic distribution as they are transported in lymph to reach the lymph nodes. This can result in longer persistence and greater antigen levels in draining lymph nodes that may lead to better immune response and reduced toxicity [10,32].
Thus, PNP-based vaccine candidates displaying viral antigenic subunits can result in more efficient interaction with B-cell receptors, a crucial step in B cell-induced immune responses that are multitudes of magnitude higher than traditional vaccines. The suitability of PNP-based vaccines for large scale production and ease of purification also adds to the growing interest in this approach. Since PNPs are often assembled from one or two protein subunits, it is also possible to generate PNP-based vaccine candidates carrying a single antigenic determinant from one or multiple pathogens or antigens from multiple strains of a single pathogen. Such mosaic PNP-based vaccine candidates offer significant advantages over conventional candidates as the need to prepare individual vaccines for each of the pathogens or strains can be avoided. As discussed above, PNP-based vaccine platforms offer many advantages for enhanced vaccine efficacy, there are also potential limitations of their use. Attaching large antigens to a PNP subunit may result in inefficient assembly of the PNP, improper orientation of the antigen, or in less ideal exposure of appropriate epitopes for protective immunological responses. Additionally, chemical conjugation or tag coupling to PNPs may result in incomplete decoration, reduced number of antigens on the PNP surface, or in conformational changes of the antigen, all of which would impact vaccine efficacy. Chemical conjugation or tag coupling approaches also require additional steps of process optimization, assembly, and purification that can compromise the eventual yield of the desired vaccine candidate.

3. Approaches for Attaching Antigens to PNP Platforms

Several different approaches have been adopted to attach antigens to PNP platforms. In the single-component system, the antigen is genetically fused with the sequences of the NP scaffold such that a single protein product is generated that assembles into a PNP carrying or displaying the antigen. In the two-component system, the surface of the NP is functionalized through genetic tags or chemical modifications for interaction with a correspondingly functionalized antigen. Each system has its own advantages and disadvantages. While in the single-component system, the ease of designing, generating, and purifying the PNPs adds significant advantages, the steric hindrance of large antigens may preclude or inhibit assembly of the PNP for optimal immune response. In the two-component approach, while the steric hindrance is not a major constraint, the yield of tag-coupled or chemically modified PNP scaffold with the antigens could be compromised. Additionally, potentially incomplete, or uneven distribution of antigens on the PNP scaffold may also lead to variability in immune responses. However, all these approaches have been used for attaching antigens to PNP platforms.

3.a. Genetic Fusion

The simplest and widely used approach to attach an antigen to a PNP platform is by genetic fusion. As PNPs are assembled from many copies of the same protein subunits, some of which have readily accessible amino- and carboxy-termini, it is relatively easy to modify the PNPs by genetically adding antigens. For example, the amino-terminus of ferritin is exposed at the surface whereas its carboxy-terminus is sequestered in the interior cavity of the NP. It is also possible to modify subunit interfaces if it does not interfere with NP assembly. The selected or designed antigen is fused in-frame with the NP sequences. The antigen can be separated from the NP by flexible linker sequences to allow proper and efficient assembly of the PNP. In cells transfected with the plasmid encoding the antigen-NP, the expressed fusion protein is assembled into PNPs which can be purified through various biochemical methods. Challenges such as suboptimal level of expression of the fusion protein, accessibility of the antigen’s termini for fusion, inefficient assembly, and steric interference due to large size of an antigen have been appropriately addressed for most PNP platforms that use genetic fusion as an approach.

3.b. Tag Coupling

The tag-coupling approach involves adding a tag to an antigen and a catcher to the NP, expressing and purifying the two components separately, and mixing them together to generate the PNPs displaying the antigen. Typically, the tag is genetically fused to one of the termini of the antigen and the catcher (or receptor) that selectively binds to the tag is fused to one of the termini of the NP such that the catcher is expressed on its surface. Following independent expression, purification, and mixing of the two components, the catcher associates with the tag with high affinity to generate the PNP displaying the antigen on its surface. The CnaB2 adhesin domain of the fibronectin binding protein, FbaB of Streptococcus pyogenes is naturally stabilized by an isopeptide bond. The side chains of an aspartic acid in a 13-residue peptide (SpyTag) and a lysine in its 116-residue protein partner (SpyCatcher) derived from the adhesin domain undergo spontaneous amidation resulting in the formation of the covalent isopeptide bond [33]. This property has been exploited for use as a simple yet highly selective and robust approach to link antigens with PNPs. The SpyTag and SpyCatcher sequences can be fused to sequences of the antigen and PNP at either terminus. With the design of more advanced SpyTag003-SpyCatcher003 possessing higher affinity with faster reaction kinetics [34], the SpyTag-SpyCatcher has become one of the most versatile and often used system to attach an antigen to a PNP platform.
Other protein/peptide-based tag coupling systems applicable for linking antigens to PNP platforms include SnoopTag/SnoopCatcher [35], sortase [36], Barnase-Barstar [37]. The SnoopTag-SnoopCatcher is also based on isopeptide bond formation through a transamidation reaction between a lysine in a 12-residue peptide tag (SnoopTag) and an asparagine in a 112-residue cognate protein partner (SnoopCatcher) derived from an adhesin molecule from Streptococcus pneumoniae. The sortase A system involves peptide bond formation through a transpeptidation reaction between a sortase A recognition motif, LPXTG (X, any amino acid) and an oligoglycine sequence at the N-terminus of a protein, which is mediated by the enzyme sortase A from Staphylococcus aureus. The Barnase-Barnstar system, on the other hand, relies on strong noncovalent interaction between dimerization domains of barnase, a 110-residue ribonuclease and an 89-residue barnstar, a barnase inhibitor from Bacillus amyloliquefaciens. Additionally, the N- and C-termini of both barnase and barstar are away from their dimerization domains and available for fusion [38]. Although the SpyTag-Spy-Catcher system is widely used, it appears that other tag coupling systems should also work well to allow specific assembly of the two components for the generation of the designed antigen-PNP complexes.

3.c. Chemical Conjugation

Chemical conjugation of an antigen to the surface of a PNP involves treatment of the two components with crosslinking agents that generate highly stable irreversible bonds [39]. A variety of crosslinking agents that target surface-exposed cysteines, lysines, glutamates and aspartates on the antigens and PNPs can be used [7]. However, chemical conjugation approaches are non-selective, can negatively impact the antigen or PNP structure, and lead to uneven decoration of the antigen, all of which could affect immune responses. In recent years, “click chemistry” has emerged as a popular approach in a variety of applications including protein labelling and modifications [40]. Among the four major classes of click reactions [41,42], the copper-catalyzed azide-alkyne cycloaddition is widely used to link two protein components. Although these reactions are fast, highly selective, and efficient, they require additional steps of introducing the reactive functional groups into the antigen and the PNPs through incorporation of amino acid analogs and unnatural amino acids into the proteins [43].

4. Protein-Based Nanoparticles (PNPs)

PNPs occur in nature in living organisms such as bacteria, archaea, viruses, plants, insects, and mammals. They exist in different shapes including spherical, rod-shaped, disk-shaped, and in sizes ranging from 8 nm to 100 nm [8,11]. Importantly, they play many critical roles in biological processes including cellular homeostasis, storage, catalysis, protection of nucleic acids, endocytic transport [8].
Based on their source, we categorize the PNPs into three major classes: (1) The naturally occurring PNPs from nonviral sources such as ferritin from various prokaryotic and eukaryotic sources, lumazine synthase, encapsulin, E2p, and small heat shock proteins from prokaryotic sources; (2) The PNPs from viral sources, generated with capsid proteins of viruses including hepatitis B virus (HBV), cucumber mosaic virus (CuMV), capsid or coat proteins of bacteriophages such as MS2, AP205, Qβ; and (3) Computationally-designed, self-assembled PNPs based on modifications to naturally occurring proteins or de novo designed proteins such as mI3, I53-50. Figure 1 shows structures of some of the PNPs that have been used as platforms for vaccines. In the sections below, we discuss these categories of PNPs with particular emphasis on the naturally occurring PNPs as they have been widely used as platforms for vaccines.

5. Vaccine Platforms Utilizing Naturally Occurring PNPs

5.a. Ferritin

5.a.i. Structure and Function

Ferritin is ubiquitous in all domains of life. It is a cytoplasmic iron storage protein with ferroxidase activity and is assembled as a NP, which stores iron within its hollow core. It also protects cells from the toxic effects of the Fenton reaction that generates hydroxyl radicals and reactive oxygen species [44,45,46]. The ferritin family consists of three distinct subfamilies: classical ferritins (or ferritins), heme-binding bacterioferritins, and DNA-binding proteins. While classical ferritins and bacterioferritins primarily serve in iron storage functions, the DNA-binding proteins facilitate iron detoxification [47,48,49]. Twenty four subunits of ferritin are assembled into a NP with octahedral symmetry that adopts a spherical cage-like structure [49,50,51] with inner and outer diameters of 8 and 12 nm, respectively (Table 1). The monomeric ferritin consists of five α-helices with the N-terminal helix being exposed outside and the C-terminal small helix sequestered inside of the hollow cavity of the NP [52]. The tertiary structure of ferritin is conserved across species. Vertebrate ferritin consists of two distinct subunits: the heavy (H) chain with a molecular mass of ~21 kDa and the light (L) chain with a mass of ~18 kDa. However, bacterial and plant ferritins exclusively comprise of only one subunit, aligning with the H chain of vertebrates [52].
In both mammalian and insect systems, ferritin NPs consist of 24 subunits of any combination of H and L chains [53] and are secreted from cells. On the other hand, bacterial ferritin NPs are assembled with 24 identical subunits into an octahedral configuration. Ferritin NPs exhibit remarkable thermal stability and resistance to chemical degradation [50,51]. These features have prompted investigators to delve extensively into exploring the structural and biochemical properties of ferritin across diverse organisms and examining its potential for developing NP based vaccine platform that leverage the distinctive characteristics of ferritin [10,54]. Central to the utilization of ferritin as vaccine platform is its capability to display antigens on the NP surface. Additionally, it has been shown not to induce any immune response [52].

5.a.ii. Viral Vaccines with Ferritin-Based NP Platform

Ferritin NP has emerged as an excellent platform for development of viral vaccines since antigens can be displayed on the surface of the NP through multiple methods. In addition, the NPs possess excellent thermal and pH stability and can be readily assembled from monomeric subunits fused to antigens. Although ferritin from prokaryotic and eukaryotic sources have been used as platforms, vast majority of studies (Table 2) have employed ferritin from H. pylori. The viral antigens are attached to the ferritin NP scaffold predominantly by genetic fusion at its N-terminus; however, tag coupling and chemical conjugation methods have also been used in some cases. The antigen-ferritin chimeric proteins are expressed in a variety of host cells including mammalian cells, insect cells, yeast, and bacteria. The expressed proteins assembled as PNPs can be designed to be secreted from the cells or extracted and purified for further characterization and vaccination studies.
The first use of ferritin NP as a vaccine platform was showcased by the presentation of influenza virus hemagglutinin (HA) in its native trimeric conformation on the NP surface. This resulted in significantly enhanced induction of neutralizing antibodies, underscoring the potential of this vaccine candidate for heightened immune responses [55]. This approach has proven successful in creating a mosaic NP “universal influenza vaccine” containing HA from multiple subtypes, conferring heterotypic protection [56,57,58]. Studies have also shown that HA-Ferritin NPs can stimulate prolonged germinal centre activity, indicative of an amplified and enduring immune response [59]. The heightened germinal centre response correlated with the maturation of memory B cells, thus enabling accelerated and more effective immune reactions upon subsequent exposures [59].
With the advent of ferritin NP platform, significant progress was made in the development of HIV vaccines. Several vaccine candidates have now been generated using this platform (Table 2) and been tested for their efficacy in animal models. In majority of the studies, it entailed the presentation of HIV-1 envelope glycoprotein trimers on ferritin NP surface to augment immune responses. Robust humoral immune responses induced by the NP-based vaccine candidates with the involvement of germinal centres as compared to the use of soluble trimers were demonstrated [60,61,62,63,64]. Immunized animals showed a marked increase in the production of neutralizing antibodies targeting various HIV-1 strains [64]. In an interesting study, ferritin NPs were tailored to present native-like BG505 SOSIP trimers, which were designed with the goal of recruiting V3-glycan-specific B cells by enhancing the accessibility of the V3-glycan patch epitope [65]. It revealed that animals immunized with these NPs exhibited elevated antibody compared to their counterparts immunized with soluble trimers [62,64,66]. Interestingly, despite the heightened antibody response, the autologous 50% neutralization titers against the Tier-2 BG505 virus in the ferritin-immunized rabbits did not exhibit improvement [64,66]. In contrast, rabbits immunized with ferritin nanoparticles carrying the consensus sequence based ConM SOSIP trimer displayed enhanced neutralization titers compared to those immunized with soluble ConM SOSIP trimers [63].
A hepatitis C virus (HCV) ferritin NP vaccine was engineered to address the challenges posed by the virus’ genetic diversity. Since the soluble E2 envelope protein (sE2) of HCV was shown to induce broadly neutralizing antibodies against all HCV genotypes in mice and macaques [67], ferritin NP displaying the sE2 was found to elicit significantly higher neutralizing antibody levels than sE2 in mice and neutralized all HCV serotypes [68]. In recent years, ferritin NP have become the choice platform for generating vaccine candidates against many viral pathogens (Table 2). A vaccine candidate for respiratory syncytial virus (RSV) displaying key neutralizing epitopes and shielding the non-neutralizing epitopes on the pre-fusion conformation of the fusion (F) glycoprotein was shown to induced durable antibody responses in nonhuman primates, generating potent neutralizing antibodies both in vivo and in vitro [69]. Ferritin NP-based vaccine candidates were also recently developed for Zika virus (ZIKV). The envelope (E) protein domain III, which carries neutralizing epitopes was displayed on either the human heavy chain ferritin [70] or H. pylori ferritin [71]. These vaccine candidates induced robust immune responses that protected animals from lethal ZIKV challenge. Furthermore, enhanced frequencies of interferon (IFN)-γ positive CD4 and CD8 T cells were also observed, indicating the induction of both humoral and cell-mediated immune responses [71].
Using a hybrid H. pylori-bullfrog ferritin as platform, investigators displayed a functionally conserved receptor-binding domain of gp350 of Epstein Barr virus (EBV). This strategy induced potent neutralizing antibodies in both mice models and non-human primates (NHPs) that were 10- to 100-fold higher than the soluble gp350 [72]. Likewise, in a recent study, ferritin NPs displaying the fusion components of EBV, namely the glycoprotein H (gH) and gL or gH/gL and gp42 were shown to induce robust immune responses in mice and NHPs that could neutralize the virus in B cells and epithelial cells [73]. These studies underscore the potential of ferritin-based nanoparticle strategies in augmenting the efficacy of vaccines against challenging viruses like EBV.
With the onset of SARS-CoV-2 pandemic and the urgent need for vaccines, attempts were made to use the PNP-based platforms including the ferritin NP-based system to generate several vaccine candidates against the virus (Table 2), some of which have moved to clinical trials [74,75,76]. The ferritin NP-based candidates display either the full-length spike (S) protein [77,78,79,80], the full S ectodomain [81], the S ectodomain with deletion of 70 C-terminal residues [82], the receptor binding domain (RBD) [79,83,84,85,86,87,88,89,90], RBD and heptad repeat (HR) [88] or a non-glycosylated RBD immunogen generated with SPEEDesign computational approach that incorporated deep mutational scanning data [91]. Majority of these candidates were generated via tag coupling or genetic fusion approaches. In various animal models and virus challenge studies, these vaccine candidates induced robust and sustained neutralizing antibody responses and cellular immune responses that conferred protection against virus challenge.

5.b. Lumazine Synthase

5.b.i. Structure and Function

Lumazine synthase (LS), a naturally occurring protein in bacteria and archaea, fungi, and plants, is integral to riboflavin (vitamin B2) synthesis and holds significant promise in the field of vaccine design [92]. LS is composed of 60 identical subunits arranged in 12 pentameric units forming an icosahedral NP structure of 15.4 nm and 9 nm outer and inner diameters, respectively [93,94]. Interestingly, LS from Bacillus subtilis at high pH conditions can reassemble into larger NP of about 29 nm containing 180 subunits [95]. Both the N- and C-termini of LS are exposed on the surface of NP and exhibit threefold and fivefold symmetry, with the N-terminus appearing closer to the threefold apex compared to the C-terminus. This proximity of the termini to the symmetry axis has significant implications for stabilizing the presentation of trimeric or pentameric antigens in an orderly array.

5.b.ii. Viral Vaccines with Lumazine-Based NP Platform

By genetically fusing through a coiled-coil linker at the C-terminus of LS from Aquifex aeolicus, a rationally designed gp120 was shown to induce a robust humoral response in mice compared to the antigen presented without the NP platform [96]. Furthermore, the designed vaccine candidate activated the germline and mature VRC01-class B cells. Presentation of native-like trimeric HIV-1 gp120 or gp140 that display 20 spikes on LS NP surface led to robust stimulation of B cells carrying cognate VRC01 receptor [60]. The LS NP platform was also used to generate HIV-1 vaccine candidates targeting the germinal centres for robust broadly neutralizing humoral immune responses [62,97].
Building on these successes, the trimeric S protein of SARS-CoV-2 presented on the surface of LS NP was shown to elicit significantly higher neutralizing antibody response compared to the S protein without the NP platform. In similar studies, the SARS-CoV-2 RBD displayed on the surface of LS NP was demonstrated to induce potent and long-lasting neutralizing antibody responses that conferred near complete protection of animals against the virus challenge [91,98]. Importantly, the neutralizing antibodies generated by this vaccine candidate could neutralize not only several variants of SARS-CoV-2 but also SARS-CoV-1 and its related bat coronaviruses [98]. In a different approach, SARS-CoV-2 RBD was linked to LS NP from Brucella abortus via sortase A-mediated transpeptidation reaction. This yielded variable number of RBD molecules ranging from as high as 6-7 to as low as 1-2 per decamer of LS. The humoral responses in vaccinated animals were significantly higher with LS NP carrying higher number of RBD molecules compared to those with fewer RBD molecules [99]. In an interesting recent study, the LS NP platform was used to present RBD-specific nanobodies obtained from a naïve alpaca phage display library. These nanobodies on LS NP were found to bind and neutralize pseudotyped SARS-CoV-2 efficiently. The study highlights LS’s role in enhancing nanobody efficacy against SARS-CoV-2 variants [100].
The LS NP platform has been used in recent years to develop other viral vaccine candidates including MERS-CoV RBD [101], Powassan virus envelope protein domain III [102], pseudorabies virus glycoprotein D [103], IAV mini-HA, and extracellular domain of M2 [104,105] EBV gp350 [106], and the head domain (Gn,, a target of neutralizing antibodies) of Rift Valley fever virus glycoprotein [107]. Genetic fusion or tag coupling was used to link the viral antigens to the NP scaffold. Vaccination with these candidates has elicited robust and high-quality antibody responses, including effective neutralization and establishment of mucosal immunity compared to monomeric antigen, and conferred protection in animal models, thus establishing that LS is a desirable PNP platform for development of vaccine candidates against viral pathogens.

5.c. Encapsulin

5.c.i. Structure and Function

Encapsulins are widely distributed in bacteria and archaea and play important roles in iron storage and mineralization, oxidative and nitrosative stress resistance, and anaerobic ammonium oxidation [6,108]. Recently, they have been identified as a class of prokaryotic nanocompartments (NCs) or NPs and have garnered significant interest in various fields of biology, including biomedicine and nanotechnology. Encapsulin self-assembles into NCs generating icosahedral structures with diameters ranging from 25 to 42 nm. Their remarkable ability to encapsulate cargo proteins with specific carboxy-terminal residues that bind to internal surface of the NCs makes them particularly intriguing for NP vaccine design [108,109]. Encapsulin also contains a flexible loop on the surface of the NCs/NPs which can be exploited to insert peptides or antigens for their surface display [5]. Encapsulin NCs exhibit variability in size and subunit composition depending on their origin. For instance, Pyrococcus furiosus encapsulin NC is made up of 180 protomers [110] with a diameter of 30-32 nm. In contrast, encapsulin NC from Thermotoga maritima consists of 60 protomers, measuring 24 nm in diameter [109], while that of Quasibacillus thermotolerance can assemble with up to 240 protomers, resulting in a larger diameter of 42 nm [109].

5.c.ii. Viral Vaccines with Encapsulin-Based NP Platform

Because of the presence of a flexible loop on the surface and its cargo loading ability, Encapsulin NCs/NPs can be used for simultaneous display of an antigen on the surface as well as incorporation of a second antigen into interior of the cavity for rational design of vaccines. In a proof-of-concept study, the ectodomain of the M2 protein of IAV was inserted in the surface loop and GFP was fused to the carboxy-terminus of encapsuling from T. maritima [111]. This vaccine design resulted in elicitation of antibody responses to both antigens in vaccinated animals. In another study, an encapsulin NC displaying on its surface a conserved HA stem domain of IAV was shown to confer protection against related IAV strains [112]. To enhance vaccine effectiveness against EBV, the domains I, II, and II of gp350 was fused at the C-terminus of encapsulin enabling the antigen on the NP surface [72]. The encapsulin NP vaccine induced potent neutralizing antibodies in mice and NHPs that were up to 100-fold higher than the soluble gp350 [72]. To use this NP platform for SARS-CoV-2 vaccine development, an encapsulin-RBD was designed, which exhibited exceptional antigenicity and long-term stability. In mouse models, the vaccine elicited robust neutralizing antibody responses following two immunizations, effectively neutralizing both the wild-type virus and its alpha, beta, and delta VOCs [113]. Even a single dose of the vaccine induced substantial neutralization activity against the omicron variant, despite reduced sensitivity compared to other variants. In a recent study, a stabilized version of encapsulin (EnDS) NP was created by introducing disulfide bonds between protomers [114]. Using the EnDS, RBDs of SARS-CoV-2 isolates from WA1 and BA.5 carrying RBD-stabilizing mutations were displayed via spyTag/SpyCatcher system to generate individual or mosaic NPs. The NPs were shown to induce significantly higher homologous and heterologous neutralizing titers [115] in mice. Interestingly, the study revealed that these vaccines can also elicit higher neutralizing antibody titers against other β-coronaviruses [115].

5.d. Viral Vaccines with Other NP Platform

Several other PNPs have also been use as vaccine platforms in recent years. The E2p (dihydrolipoamide acetyltransferase) from Geobacillus stearothermophilus, a component of the pyruvate dehydrogenase complex, assembles as a 27 nm NP with icosahedral symmetry consisting of 60 subunits [116,117]. The E2P NPs displaying variety of antigens including B and T cell epitopes of HIV-1 antigens through genetic fusion have resulted in induction of humoral as well as potent helper and cytotoxic T cell responses [118,119,120,121]. Recently, presentation of stabilized gp140 trimer on the surface of E2p NP led to stimulation of B cells carrying the cognate receptor [60]. Ebola virus GP rationally designed to form trimers when presented on the surface of E2p elicited vaccine-induced B-cell responses and generated cross-ebolavirus neutralizing antibodies, suggesting a promising vaccine strategy for filoviruses [122]. When an enhanced SARS-CoV-2 RBD immunogen was displayed on E2p NP, elicitation of potent neutralizing antibodies was demonstrated in NHPs [91]. Although E2p NP has not been as widely used as the other NPs discussed above, it holds significant promise since foreign antigens fused to the N-terminus of the E2p core can be readily displayed on the NP surface for immune responses. Another interesting protein, the small heat shock protein (sHSP) from Methanococcus jannaschii, which confers thermotolerance to the organism. Twenty four subunits of sHSP are assembled into a hollow spherical NP of 12 nm in outer and 6.5 nm inner diameter [123]. Interestingly, mice treated with sHSP NP alone (without any specific viral antigen) were protected against several respiratory viruses [124], indicating that the NP platform itself confers protection. Furthermore, the exterior and interior of the NP can be modified by genetic and chemical methods [125], suggesting that sHSP NP can be a highly versatile platform for vaccines.

6. Vaccine Platforms Utilizing PNPs Consisting of Viral Capsid/Coat Proteins

The capsid proteins of many viruses such as hepatitis B virus (HBV), cucumber mosaic virus (CuMV), cowpea chlorotic mottle virus and the coat proteins of bacteriophages such as AP205, MS2, Qβ are assembled into PNPs having various sizes and symmetry (Table 1) in the absence of their nucleic acid genome. These PNPs can also be readily functionalized by genetic fusion, tag coupling, or by chemical modifications to display foreign peptides or proteins on their surface without compromising the PNP assembling capabilities. These attributes have been exploited in recent years for their use as vaccine platforms. The hepatitis B virus core antigen (HBcAg) is assembled as two different types of icosahedral particles consisting of 180 (90 dimers; 26 nm; T=3) or 240 (120 dimers; 30 nm; T=4) subunits [126]. Foreign proteins can be displayed on HBcAg PNP surface readily by genetic fusion at the major immunodominant epitope (MIE), the N- or the C-termini [127,128]. The E protein domain III of ZIKV (zE-DIII), when displayed on the surface of HBcAg through genetic fusion at its C-terminus, induced robust humoral and cellular immune responses in mice, and conferred protection from multiple ZIKV strains. HBcAg PNP appears to be a very exciting platform as it can be genetically modified at many locations for surface display of an antigen and the observations that is known to contain strong T-cell epitopes [129].
Another platform that has gained significant attention is the cauliflower mosaic virus capsid PNP [130] incorporating a universal T-cell epitope from tetanus toxin (CuMVTT) [131]. Using this platform, the ZIKV E-DIII, displayed on the surface of the NP through chemical crosslinking was shown to elicit neutralizing antibody response [132]. The same platform was used generate several SARS-CoV-2 vaccine candidates using the RBD [133,134], receptor binding motif alone (RBM) [135], or along with fusion peptide [136]. The antigens were either genetically fused or chemically crosslinked to the PNP platform. These studies revealed that the antigens are highly immunogenic in animals, induce neutralizing antibody responses, cross-reactive, and can potently neutralize the virus under in vitro conditions.
In addition to the viral capsid PNPs, the coat proteins of bacteriophages that generate highly ordered spherical structures with differing triangulation numbers and symmetry have also been used as platforms for vaccine development. The Qβ VLPs [137] when linked to the extracellular domain of M2 protein of influenza virus, the resulting VLP induced strong M2-specific antibody responses and protected animals against lethal challenge with influenza virus [138]. The coat protein of bacteriophage AP205 [139] can generate stable PNPs and its N- and C-termini can accept insertions of antigens without adverse effects on PNP integrity or stability [140]. Tag coupling can also be used to display antigens on the PNP surface. This platform was used to display trimeric envelope protein of HIV-1 via the SpyTag/SpyCatcher coupling. Immunization of mice, rabbits, and NHPs led to elicitation of broadly neutralizing antibodies [141]. A similar approach was used to generate vaccine candidate for IAV by conjugating up to eight different homotypic or heterotypic (mosaic) trimers of HAs via SpyTag/SpyCatcher system. Immunization of mice with these vaccines resulted in cross-reactive antibody responses [142]. The SARS-CoV-2 RBM or RBD was displayed on the surface of the AP205 coat protein PNPs through genetic fusion at its C-terminus [143] or via SpyTag/SpyCatcher coupling [144]. Mice administered with these vaccine candidates induced elevated levels of serum antibodies and significant levels of neutralizing antibody titers. These studies suggest that AP205 coat protein PNP platform is a promising platform for vaccine development. The capsid of the bacteriophage MS2 has an icosahedral symmetry with 180 subunits of its coat protein arranged as 90 homodimers. A genetically fused single-chain dimer of two identical coat proteins can also assemble efficiently and can accommodate peptides in a surface exposed loop region [145]. Taking advantage of this, the full-length S protein of SARS-CoV-2 was displayed on the surface of the PNP by biotin-streptavidin tag coupling [146]. Following immunization, the nanoparticles were shown to generate high titers of neutralizing antibody and protected animals from SARS-CoV-2 challenge, suggesting that this platform could be used for other pathogens.

7. Vaccine Platforms Utilizing Computationally Designed PNPs

Computationally designed NP assemblies have gained significant advancement since the development of Rosetta3 for modelling symmetrical protein structures [147]. This has led to the first design of a highly sTable 25 nm size icosahedral protein structure (mI3) from 60 subunits of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from T. maritima [148]. Subsequent studies led to the design and characterization of a novel icosahedral particle (I53-50) of 30 nm consisting of 20 trimeric (A component) and twelve pentameric (B component) building blocks for a total of 120 subunits [149]. This I53-50 PNP has become the preferred choice as a platform for vaccine development.
Using the I53-50 platform, a promising vaccine targeting the fusion (F) protein of respiratory syncytial virus was developed. Traditional vaccine candidates focused on the more stable post-fusion structure due to the instability of the prefusion conformation, yielding limited efficacy in clinical trials [150,151,152]. However, employing a structure-guided approach, a self-assembling PNP displaying 20 copies of trimeric DS-Cav1, a prefusion-stabilized F protein variant was generated. This PNP vaccine induced 10-fold greater neutralizing antibody titers compared to soluble DS-Cav1 in mice and NHPs [153], demonstrating the significant potential of this I53-50 PNP platform for other viral pathogens with trimeric surface glycoprotein targets. Using a similar approach, the HA trimers of the licensed quadrivalent influenza vaccines displayed on the I53-50 platform induced broadly protective antibody responses at similar or higher levels than the licensed quadrivalent vaccines [154].
The I53-50 PNP platform was employed to enhance the immunogenicity of HIV-1 envelope trimers with the goal of inducing broadly neutralizing antibodies that can neutralize both neutralization- sensitive and -resistant viruses. To achieve this, a soluble HIV-1 envelope trimer, stabilized SOSIP trimers, expressing multiple epitopes for broadly neutralizing activity [155], was presented as 20 trimers on the surface of I53-50 PNP. This PNP vaccine candidate was shown to increase immunogenicity and enhance the quality of antibody response upon immunization [156], indicating that the I53-50 PNP would be a suitable platform for advancing efficacious HIV vaccines development. In line with these goals, further studies using this platform revealed that that such a vaccine candidate could generate substantial enhancement of neutralizing antibody titers compared to the soluble SOSIP trimers in rabbit immunization experiments [157]. Further, the neutralizing responses were directed to an immunodominant epitope, suggesting that this PNP has potential as an excellent platform for HIV vaccine development.
With the onset of SARS-CoV-2 pandemic, the I53-50 PNP became the platform of choice to quickly generate a variety of vaccine candidates against the virus. The ease with which the two components of I53-50 could be readily constructed, produced, purified, and assembled into PNPs displaying the SARS-CoV-2 antigenic determinants sped up the development of several excellent vaccine candidates. In these studies, either the full-length S [158], mosaic S from several variants of concern [159], the RBD [76,160] or the mosaic RBD from multiple sarbecoviruses [161] was displayed as trimeric subunits on the surface of the I53-50 through genetic fusion or tag coupling. In various animal models, these vaccine candidates induced significantly higher neutralizing antibody responses compared to their soluble trimeric counterparts. These studies suggest that I53-50 is an excellent antigen display platform for vaccine development.

8. Conclusions

As emerging and re-emerging viral and other infectious agents pose serious health challenges worldwide, rapid development of safe, highly efficacious, and cost-effective vaccines is needed. PNP-based vaccine platforms offer significant advantages in this regard. These platforms are highly reproducible, scalable, the target antigen can be attached readily through a variety of approaches without significantly compromising its native-like structure, and exhibit adjuvant-like properties for enhanced immune responses and protection. In recent years, many PNPs have been used as excellent platforms for displaying viral antigens and such vaccine candidates have unequivocally shown to induce significantly elevated immune responses resulting in protection from the agent. With the ability to computationally design and experimentally characterize icosahedral PNPs, it will be possible to further optimize these structures with regard to their primary sequence, subunit interaction, assembly, and presentation of antigens. Such optimizations would undoubtedly lead to candidate vaccines with unprecedented safely and efficacy.

Author Contributions

Conceptualization: KKP; writing-original draft preparation: KKP, BRS; writing-review and editing: KKP, BRS, and AKP; supervision: AKP; project administration: AKP; funding acquisition: AKP. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by funds from the University of Nebraska-Lincoln

Conflicts of Interests

The authors declare no conflicts of interest.

References

  1. Karch, C.P.; Burkhard, P. Vaccine technologies: From whole organisms to rationally designed protein assemblies. Biochem Pharmacol 2016, 120, 1–14. [Google Scholar] [CrossRef] [PubMed]
  2. Delrue, I.; Verzele, D.; Madder, A.; Nauwynck, H.J. Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges. Expert Rev Vaccines 2012, 11, 695–719. [Google Scholar] [CrossRef] [PubMed]
  3. Moon, J.J.; Suh, H.; Li, A.V.; Ockenhouse, C.F.; Yadava, A.; Irvine, D.J. Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction. Proc Natl Acad Sci U S A 2012, 109, 1080–1085. [Google Scholar] [CrossRef] [PubMed]
  4. Yen, H.J.; Hsu, S.H.; Tsai, C.L. Cytotoxicity and immunological response of gold and silver nanoparticles of different sizes. Small 2009, 5, 1553–1561. [Google Scholar] [CrossRef]
  5. Obozina, A.S.; Komedchikova, E.N.; Kolesnikova, O.A.; Iureva, A.M.; Kovalenko, V.L.; Zavalko, F.A.; Rozhnikova, T.V.; Tereshina, E.D.; Mochalova, E.N.; Shipunova, V.O. Genetically Encoded Self-Assembling Protein Nanoparticles for the Targeted Delivery In Vitro and In Vivo. Pharmaceutics 2023, 15. [Google Scholar] [CrossRef] [PubMed]
  6. Giessen, T.W.; Silver, P.A. Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat Microbiol 2017, 2, 17029. [Google Scholar] [CrossRef] [PubMed]
  7. Schoonen, L.; van Hest, J.C. Functionalization of protein-based nanocages for drug delivery applications. Nanoscale 2014, 6, 7124–7141. [Google Scholar] [CrossRef] [PubMed]
  8. Lee, E.J. Recent advances in protein-based nanoparticles. Korean Journal of Chemical Engineering 2018, 35, 1765–1778. [Google Scholar] [CrossRef]
  9. Diaz, D.; Care, A.; Sunna, A. Bioengineering Strategies for Protein-Based Nanoparticles. Genes (Basel) 2018, 9. [Google Scholar] [CrossRef]
  10. Lamontagne, F.; Khatri, V.; St-Louis, P.; Bourgault, S.; Archambault, D. Vaccination Strategies Based on Bacterial Self-Assembling Proteins as Antigen Delivery Nanoscaffolds. Vaccines (Basel) 2022, 10. [Google Scholar] [CrossRef]
  11. Pieters, B.J.; van Eldijk, M.B.; Nolte, R.J.; Mecinovic, J. Natural supramolecular protein assemblies. Chem Soc Rev 2016, 45, 24–39. [Google Scholar] [CrossRef] [PubMed]
  12. Douglas, T.; Young, M. Viruses: making friends with old foes. Science 2006, 312, 873–875. [Google Scholar] [CrossRef] [PubMed]
  13. Bachmann, M.F.; Jennings, G.T. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol 2010, 10, 787–796. [Google Scholar] [CrossRef] [PubMed]
  14. Roth, G.A.; Picece, V.; Ou, B.S.; Luo, W.; Pulendran, B.; Appel, E.A. Designing spatial and temporal control of vaccine responses. Nat Rev Mater 2022, 7, 174–195. [Google Scholar] [CrossRef] [PubMed]
  15. Ke, X.; Howard, G.P.; Tang, H.; Cheng, B.; Saung, M.T.; Santos, J.L.; Mao, H.Q. Physical and chemical profiles of nanoparticles for lymphatic targeting. Adv Drug Deliv Rev 2019, 151-152, 72–93. [Google Scholar] [CrossRef] [PubMed]
  16. Irvine, D.J.; Read, B.J. Shaping humoral immunity to vaccines through antigen-displaying nanoparticles. Curr Opin Immunol 2020, 65, 1–6. [Google Scholar] [CrossRef] [PubMed]
  17. Link, A.; Zabel, F.; Schnetzler, Y.; Titz, A.; Brombacher, F.; Bachmann, M.F. Innate immunity mediates follicular transport of particulate but not soluble protein antigen. J Immunol 2012, 188, 3724–3733. [Google Scholar] [CrossRef]
  18. Gause, K.T.; Wheatley, A.K.; Cui, J.; Yan, Y.; Kent, S.J.; Caruso, F. Immunological Principles Guiding the Rational Design of Particles for Vaccine Delivery. ACS Nano 2017, 11, 54–68. [Google Scholar] [CrossRef] [PubMed]
  19. Kelly, H.G.; Kent, S.J.; Wheatley, A.K. Immunological basis for enhanced immunity of nanoparticle vaccines. Expert Rev Vaccines 2019, 18, 269–280. [Google Scholar] [CrossRef]
  20. Slifka, M.K.; Amanna, I.J. Role of Multivalency and Antigenic Threshold in Generating Protective Antibody Responses. Front Immunol 2019, 10, 956. [Google Scholar] [CrossRef]
  21. Zabel, F.; Mohanan, D.; Bessa, J.; Link, A.; Fettelschoss, A.; Saudan, P.; Kundig, T.M.; Bachmann, M.F. Viral particles drive rapid differentiation of memory B cells into secondary plasma cells producing increased levels of antibodies. J Immunol 2014, 192, 5499–5508. [Google Scholar] [CrossRef] [PubMed]
  22. Lopez-Sagaseta, J.; Malito, E.; Rappuoli, R.; Bottomley, M.J. Self-assembling protein nanoparticles in the design of vaccines. Comput Struct Biotechnol J 2016, 14, 58–68. [Google Scholar] [CrossRef] [PubMed]
  23. Kato, Y.; Abbott, R.K.; Freeman, B.L.; Haupt, S.; Groschel, B.; Silva, M.; Menis, S.; Irvine, D.J.; Schief, W.R.; Crotty, S. Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo. Immunity 2020, 53, 548–563. [Google Scholar] [CrossRef] [PubMed]
  24. Foged, C.; Brodin, B.; Frokjaer, S.; Sundblad, A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm 2005, 298, 315–322. [Google Scholar] [CrossRef]
  25. Reddy, S.T.; Swartz, M.A.; Hubbell, J.A. Targeting dendritic cells with biomaterials: developing the next generation of vaccines. Trends Immunol 2006, 27, 573–579. [Google Scholar] [CrossRef] [PubMed]
  26. Lung, P.; Yang, J.; Li, Q. Nanoparticle formulated vaccines: opportunities and challenges. Nanoscale 2020, 12, 5746–5763. [Google Scholar] [CrossRef] [PubMed]
  27. Karch, C.P.; Li, J.; Kulangara, C.; Paulillo, S.M.; Raman, S.K.; Emadi, S.; Tan, A.; Helal, Z.H.; Fan, Q.; Khan, M.I.; et al. Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge. Nanomedicine 2017, 13, 241–251. [Google Scholar] [CrossRef] [PubMed]
  28. Lynn, G.M.; Sedlik, C.; Baharom, F.; Zhu, Y.; Ramirez-Valdez, R.A.; Coble, V.L.; Tobin, K.; Nichols, S.R.; Itzkowitz, Y.; Zaidi, N.; et al. Peptide-TLR-7/8a conjugate vaccines chemically programmed for nanoparticle self-assembly enhance CD8 T-cell immunity to tumor antigens. Nat Biotechnol 2020, 38, 320–332. [Google Scholar] [CrossRef]
  29. Qiao, Y.; Zhang, Y.; Chen, J.; Jin, S.; Shan, Y. A biepitope, adjuvant-free, self-assembled influenza nanovaccine provides cross-protection against H3N2 and H1N1 viruses in mice. Nano Res 2022, 15, 8304–8314. [Google Scholar] [CrossRef]
  30. Nie, J.; Zhou, Y.; Ding, F.; Liu, X.; Yao, X.; Xu, L.; Chang, Y.; Li, Z.; Wang, Q.; Zhan, L.; et al. Self-adjuvant multiepitope nanovaccine based on ferritin induced long-lasting and effective mucosal immunity against H3N2 and H1N1 viruses in mice. Int J Biol Macromol 2024, 259, 129259. [Google Scholar] [CrossRef]
  31. Rossi, A.H.; Farias, A.; Fernandez, J.E.; Bonomi, H.R.; Goldbaum, F.A.; Berguer, P.M. Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response against B16 Melanoma in Mice. PLoS One 2015, 10, e0126827. [Google Scholar] [CrossRef]
  32. Irvine, D.J.; Aung, A.; Silva, M. Controlling timing and location in vaccines. Adv Drug Deliv Rev 2020, 158, 91–115. [Google Scholar] [CrossRef]
  33. Zakeri, B.; Fierer, J.O.; Celik, E.; Chittock, E.C.; Schwarz-Linek, U.; Moy, V.T.; Howarth, M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 2012, 109, E690–E697. [Google Scholar] [CrossRef]
  34. Keeble, A.H.; Turkki, P.; Stokes, S.; Khairil Anuar, I.N.A.; Rahikainen, R.; Hytonen, V.P.; Howarth, M. Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A 2019, 116, 26523–26533. [Google Scholar] [CrossRef] [PubMed]
  35. Veggiani, G.; Nakamura, T.; Brenner, M.D.; Gayet, R.V.; Yan, J.; Robinson, C.V.; Howarth, M. Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A 2016, 113, 1202–1207. [Google Scholar] [CrossRef] [PubMed]
  36. Guimaraes, C.P.; Witte, M.D.; Theile, C.S.; Bozkurt, G.; Kundrat, L.; Blom, A.E.; Ploegh, H.L. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat Protoc 2013, 8, 1787–1799. [Google Scholar] [CrossRef] [PubMed]
  37. Deyev, S.M.; Waibel, R.; Lebedenko, E.N.; Schubiger, A.P.; Pluckthun, A. Design of multivalent complexes using the barnase*barstar module. Nat Biotechnol 2003, 21, 1486–1492. [Google Scholar] [CrossRef]
  38. Buckle, A.M.; Schreiber, G.; Fersht, A.R. Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution. Biochemistry 1994, 33, 8878–8889. [Google Scholar] [CrossRef]
  39. Liebana, S.; Drago, G.A. Bioconjugation and stabilisation of biomolecules in biosensors. Essays Biochem 2016, 60, 59–68. [Google Scholar] [CrossRef]
  40. Yao, T.; Xu, X.; Huang, R. Recent Advances about the Applications of Click Reaction in Chemical Proteomics. Molecules 2021, 26. [Google Scholar] [CrossRef]
  41. Hein, C.D.; Liu, X.M.; Wang, D. Click chemistry, a powerful tool for pharmaceutical sciences. Pharm Res 2008, 25, 2216–2230. [Google Scholar] [CrossRef] [PubMed]
  42. Huisgen, R. 1,3-Dipolar Cycloadditions. Past and Future†. Angewandte Chemie 1963, 2, 565–598. [Google Scholar] [CrossRef]
  43. Smith, M.T.; Hawes, A.K.; Bundy, B.C. Reengineering viruses and virus-like particles through chemical functionalization strategies. Curr Opin Biotechnol 2013, 24, 620–626. [Google Scholar] [CrossRef] [PubMed]
  44. Pantopoulos, K.; Porwal, S.K.; Tartakoff, A.; Devireddy, L. Mechanisms of mammalian iron homeostasis. Biochemistry 2012, 51, 5705–5724. [Google Scholar] [CrossRef] [PubMed]
  45. Andrews, S.C. The Ferritin-like superfamily: Evolution of the biological iron storeman from a rubrerythrin-like ancestor. Biochim Biophys Acta 2010, 1800, 691–705. [Google Scholar] [CrossRef] [PubMed]
  46. Chasteen, N.D.; Harrison, P.M. Mineralization in ferritin: an efficient means of iron storage. J Struct Biol 1999, 126, 182–194. [Google Scholar] [CrossRef] [PubMed]
  47. Andrews, S.C. Iron storage in bacteria. Adv Microb Physiol 1998, 40, 281–351. [Google Scholar] [CrossRef]
  48. Andrews, S.C.; Robinson, A.K.; Rodríguez-Quiñones, F. Bacterial iron homeostasis. FEMS Microbiol Rev 2003, 27, 215–237. [Google Scholar] [CrossRef]
  49. Arosio, P.; Elia, L.; Poli, M. Ferritin, cellular iron storage and regulation. IUBMB Life 2017, 69, 414–422. [Google Scholar] [CrossRef]
  50. Cho, K.J.; Shin, H.J.; Lee, J.H.; Kim, K.J.; Park, S.S.; Lee, Y.; Lee, C.; Park, S.S.; Kim, K.H. The crystal structure of ferritin from Helicobacter pylori reveals unusual conformational changes for iron uptake. J Mol Biol 2009, 390, 83–98. [Google Scholar] [CrossRef]
  51. He, D.; Marles-Wright, J. Ferritin family proteins and their use in bionanotechnology. N Biotechnol 2015, 32, 651–657. [Google Scholar] [CrossRef]
  52. Harrison, P.M.; Arosio, P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta 1996, 1275, 161–203. [Google Scholar] [CrossRef] [PubMed]
  53. Hamburger, A.E.; West, A.P., Jr.; Hamburger, Z.A.; Hamburger, P.; Bjorkman, P.J. Crystal structure of a secreted insect ferritin reveals a symmetrical arrangement of heavy and light chains. J Mol Biol 2005, 349, 558–569. [Google Scholar] [CrossRef] [PubMed]
  54. Rodrigues, M.Q.; Alves, P.M.; Roldao, A. Functionalizing Ferritin Nanoparticles for Vaccine Development. Pharmaceutics 2021, 13. [Google Scholar] [CrossRef]
  55. Kanekiyo, M.; Wei, C.J.; Yassine, H.M.; McTamney, P.M.; Boyington, J.C.; Whittle, J.R.; Rao, S.S.; Kong, W.P.; Wang, L.; Nabel, G.J. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 2013, 499, 102–106. [Google Scholar] [CrossRef] [PubMed]
  56. Yassine, H.M.; Boyington, J.C.; McTamney, P.M.; Wei, C.J.; Kanekiyo, M.; Kong, W.P.; Gallagher, J.R.; Wang, L.; Zhang, Y.; Joyce, M.G.; et al. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nat Med 2015, 21, 1065–1070. [Google Scholar] [CrossRef]
  57. Darricarrere, N.; Pougatcheva, S.; Duan, X.; Rudicell, R.S.; Chou, T.H.; DiNapoli, J.; Ross, T.M.; Alefantis, T.; Vogel, T.U.; Kleanthous, H.; et al. Development of a Pan-H1 Influenza Vaccine. J Virol 2018, 92. [Google Scholar] [CrossRef]
  58. Kanekiyo, M.; Joyce, M.G.; Gillespie, R.A.; Gallagher, J.R.; Andrews, S.F.; Yassine, H.M.; Wheatley, A.K.; Fisher, B.E.; Ambrozak, D.R.; Creanga, A.; et al. Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses. Nat Immunol 2019, 20, 362–372. [Google Scholar] [CrossRef]
  59. Kelly, H.G.; Tan, H.X.; Juno, J.A.; Esterbauer, R.; Ju, Y.; Jiang, W.; Wimmer, V.C.; Duckworth, B.C.; Groom, J.R.; Caruso, F.; et al. Self-assembling influenza nanoparticle vaccines drive extended germinal center activity and memory B cell maturation. JCI Insight 2020, 5. [Google Scholar] [CrossRef]
  60. He, L.; de Val, N.; Morris, C.D.; Vora, N.; Thinnes, T.C.; Kong, L.; Azadnia, P.; Sok, D.; Zhou, B.; Burton, D.R.; et al. Presenting native-like trimeric HIV-1 antigens with self-assembling nanoparticles. Nat Commun 2016, 7, 12041. [Google Scholar] [CrossRef]
  61. Sliepen, K.; Schermer, E.; Bontjer, I.; Burger, J.A.; Levai, R.F.; Mundsperger, P.; Brouwer, P.J.M.; Tolazzi, M.; Farsang, A.; Katinger, D.; et al. Interplay of diverse adjuvants and nanoparticle presentation of native-like HIV-1 envelope trimers. NPJ Vaccines 2021, 6, 103. [Google Scholar] [CrossRef] [PubMed]
  62. Tokatlian, T.; Read, B.J.; Jones, C.A.; Kulp, D.W.; Menis, S.; Chang, J.Y.H.; Steichen, J.M.; Kumari, S.; Allen, J.D.; Dane, E.L.; et al. Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers. Science 2019, 363, 649–654. [Google Scholar] [CrossRef]
  63. Sliepen, K.; Han, B.W.; Bontjer, I.; Mooij, P.; Garces, F.; Behrens, A.J.; Rantalainen, K.; Kumar, S.; Sarkar, A.; Brouwer, P.J.M.; et al. Structure and immunogenicity of a stabilized HIV-1 envelope trimer based on a group-M consensus sequence. Nat Commun 2019, 10, 2355. [Google Scholar] [CrossRef] [PubMed]
  64. Sliepen, K.; Ozorowski, G.; Burger, J.A.; van Montfort, T.; Stunnenberg, M.; LaBranche, C.; Montefiori, D.C.; Moore, J.P.; Ward, A.B.; Sanders, R.W. Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity. Retrovirology 2015, 12, 82. [Google Scholar] [CrossRef] [PubMed]
  65. Bianchi, M.; Turner, H.L.; Nogal, B.; Cottrell, C.A.; Oyen, D.; Pauthner, M.; Bastidas, R.; Nedellec, R.; McCoy, L.E.; Wilson, I.A.; et al. Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization. Immunity 2018, 49, 288–300. [Google Scholar] [CrossRef] [PubMed]
  66. He, L.; Kumar, S.; Allen, J.D.; Huang, D.; Lin, X.; Mann, C.J.; Saye-Francisco, K.L.; Copps, J.; Sarkar, A.; Blizard, G.S.; et al. HIV-1 vaccine design through minimizing envelope metastability. Sci Adv 2018, 4, eaau6769. [Google Scholar] [CrossRef]
  67. Li, D.; Wang, X.; von Schaewen, M.; Tao, W.; Zhang, Y.; Heller, B.; Hrebikova, G.; Deng, Q.; Sun, Q.; Ploss, A.; et al. Immunization With a Subunit Hepatitis C Virus Vaccine Elicits Pan-Genotypic Neutralizing Antibodies and Intrahepatic T-Cell Responses in Nonhuman Primates. J Infect Dis 2017, 215, 1824–1831. [Google Scholar] [CrossRef] [PubMed]
  68. Yan, Y.; Wang, X.; Lou, P.; Hu, Z.; Qu, P.; Li, D.; Li, Q.; Xu, Y.; Niu, J.; He, Y.; et al. A Nanoparticle-Based Hepatitis C Virus Vaccine With Enhanced Potency. J Infect Dis 2020, 221, 1304–1314. [Google Scholar] [CrossRef]
  69. Swanson, K.A.; Rainho-Tomko, J.N.; Williams, Z.P.; Lanza, L.; Peredelchuk, M.; Kishko, M.; Pavot, V.; Alamares-Sapuay, J.; Adhikarla, H.; Gupta, S.; et al. A respiratory syncytial virus (RSV) F protein nanoparticle vaccine focuses antibody responses to a conserved neutralization domain. Sci Immunol 2020, 5. [Google Scholar] [CrossRef]
  70. Rong, H.; Qi, M.; Pan, J.; Sun, Y.; Gao, J.; Zhang, X.; Li, W.; Zhang, B.; Zhang, X.E.; Cui, Z. Self-Assembling Nanovaccine Confers Complete Protection Against Zika Virus Without Causing Antibody-Dependent Enhancement. Front Immunol 2022, 13, 905431. [Google Scholar] [CrossRef]
  71. Pattnaik, A.; Sahoo, B.R.; Struble, L.R.; Borgstahl, G.E.O.; Zhou, Y.; Franco, R.; Barletta, R.G.; Osorio, F.A.; Petro, T.M.; Pattnaik, A.K. A Ferritin Nanoparticle-Based Zika Virus Vaccine Candidate Induces Robust Humoral and Cellular Immune Responses and Protects Mice from Lethal Virus Challenge. Vaccines (Basel) 2023, 11. [Google Scholar] [CrossRef] [PubMed]
  72. Kanekiyo, M.; Bu, W.; Joyce, M.G.; Meng, G.; Whittle, J.R.; Baxa, U.; Yamamoto, T.; Narpala, S.; Todd, J.P.; Rao, S.S.; et al. Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site. Cell 2015, 162, 1090–1100. [Google Scholar] [CrossRef] [PubMed]
  73. Wei, C.J.; Bu, W.; Nguyen, L.A.; Batchelor, J.D.; Kim, J.; Pittaluga, S.; Fuller, J.R.; Nguyen, H.; Chou, T.H.; Cohen, J.I.; et al. A bivalent Epstein-Barr virus vaccine induces neutralizing antibodies that block infection and confer immunity in humanized mice. Sci Transl Med 2022, 14, eabf3685. [Google Scholar] [CrossRef] [PubMed]
  74. King, H.A.D.; Joyce, M.G.; Lakhal-Naouar, I.; Ahmed, A.; Cincotta, C.M.; Subra, C.; Peachman, K.K.; Hack, H.R.; Chen, R.E.; Thomas, P.V.; et al. Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques. Proc Natl Acad Sci U S A 2021, 118. [Google Scholar] [CrossRef]
  75. Joyce, M.G.; Chen, W.H.; Sankhala, R.S.; Hajduczki, A.; Thomas, P.V.; Choe, M.; Martinez, E.J.; Chang, W.C.; Peterson, C.E.; Morrison, E.B.; et al. SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity. Cell Rep 2021, 37, 110143. [Google Scholar] [CrossRef] [PubMed]
  76. Walls, A.C.; Fiala, B.; Schafer, A.; Wrenn, S.; Pham, M.N.; Murphy, M.; Tse, L.V.; Shehata, L.; O’Connor, M.A.; Chen, C.; et al. Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2. Cell 2020, 183, 1367–1382. [Google Scholar] [CrossRef] [PubMed]
  77. Carmen, J.M.; Shrivastava, S.; Lu, Z.; Anderson, A.; Morrison, E.B.; Sankhala, R.S.; Chen, W.H.; Chang, W.C.; Bolton, J.S.; Matyas, G.R.; et al. SARS-CoV-2 ferritin nanoparticle vaccine induces robust innate immune activity driving polyfunctional spike-specific T cell responses. NPJ Vaccines 2021, 6, 151. [Google Scholar] [CrossRef]
  78. Zhang, B.; Chao, C.W.; Tsybovsky, Y.; Abiona, O.M.; Hutchinson, G.B.; Moliva, J.I.; Olia, A.S.; Pegu, A.; Phung, E.; Stewart-Jones, G.B.E.; et al. A platform incorporating trimeric antigens into self-assembling nanoparticles reveals SARS-CoV-2-spike nanoparticles to elicit substantially higher neutralizing responses than spike alone. Sci Rep 2020, 10, 18149. [Google Scholar] [CrossRef] [PubMed]
  79. He, L.; Lin, X.; Wang, Y.; Abraham, C.; Sou, C.; Ngo, T.; Zhang, Y.; Wilson, I.A.; Zhu, J. Single-component, self-assembling, protein nanoparticles presenting the receptor binding domain and stabilized spike as SARS-CoV-2 vaccine candidates. Sci Adv 2021, 7. [Google Scholar] [CrossRef]
  80. Joyce, M.G.; King, H.A.D.; Elakhal-Naouar, I.; Ahmed, A.; Peachman, K.K.; Macedo Cincotta, C.; Subra, C.; Chen, R.E.; Thomas, P.V.; Chen, W.H.; et al. A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates. Sci Transl Med 2022, 14, eabi5735. [Google Scholar] [CrossRef]
  81. Powell, A.E.; Zhang, K.; Sanyal, M.; Tang, S.; Weidenbacher, P.A.; Li, S.; Pham, T.D.; Pak, J.E.; Chiu, W.; Kim, P.S. A Single Immunization with Spike-Functionalized Ferritin Vaccines Elicits Neutralizing Antibody Responses against SARS-CoV-2 in Mice. ACS Cent Sci 2021, 7, 183–199. [Google Scholar] [CrossRef] [PubMed]
  82. Weidenbacher, P.A.; Sanyal, M.; Friedland, N.; Tang, S.; Arunachalam, P.S.; Hu, M.; Kumru, O.S.; Morris, M.K.; Fontenot, J.; Shirreff, L.; et al. A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates. Nat Commun 2023, 14, 2149. [Google Scholar] [CrossRef]
  83. Salzer, R.; Clark, J.J.; Vaysburd, M.; Chang, V.T.; Albecka, A.; Kiss, L.; Sharma, P.; Gonzalez Llamazares, A.; Kipar, A.; Hiscox, J.A.; et al. Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice. FEBS Lett 2021, 595, 2323–2340. [Google Scholar] [CrossRef]
  84. Kang, Y.F.; Sun, C.; Zhuang, Z.; Yuan, R.Y.; Zheng, Q.; Li, J.P.; Zhou, P.P.; Chen, X.C.; Liu, Z.; Zhang, X.; et al. Rapid Development of SARS-CoV-2 Spike Protein Receptor-Binding Domain Self-Assembled Nanoparticle Vaccine Candidates. ACS Nano 2021, 15, 2738–2752. [Google Scholar] [CrossRef] [PubMed]
  85. Li, H.; Guo, L.; Zheng, H.; Li, J.; Zhao, X.; Li, J.; Liang, Y.; Yang, F.; Zhao, Y.; Yang, J.; et al. Self-Assembling Nanoparticle Vaccines Displaying the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Responses in Rhesus Monkeys. Bioconjug Chem 2021, 32, 1034–1046. [Google Scholar] [CrossRef] [PubMed]
  86. Kim, Y.I.; Kim, D.; Yu, K.M.; Seo, H.D.; Lee, S.A.; Casel, M.A.B.; Jang, S.G.; Kim, S.; Jung, W.; Lai, C.J.; et al. Development of Spike Receptor-Binding Domain Nanoparticles as a Vaccine Candidate against SARS-CoV-2 Infection in Ferrets. mBio 2021, 12. [Google Scholar] [CrossRef]
  87. Sun, W.; He, L.; Zhang, H.; Tian, X.; Bai, Z.; Sun, L.; Yang, L.; Jia, X.; Bi, Y.; Luo, T.; et al. The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice. Signal Transduct Target Ther 2021, 6, 340. [Google Scholar] [CrossRef]
  88. Ma, X.; Zou, F.; Yu, F.; Li, R.; Yuan, Y.; Zhang, Y.; Zhang, X.; Deng, J.; Chen, T.; Song, Z.; et al. Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses. Immunity 2020, 53, 1315–1330. [Google Scholar] [CrossRef]
  89. Wang, W.; Huang, B.; Zhu, Y.; Tan, W.; Zhu, M. Ferritin nanoparticle-based SARS-CoV-2 RBD vaccine induces a persistent antibody response and long-term memory in mice. Cell Mol Immunol 2021, 18, 749–751. [Google Scholar] [CrossRef]
  90. Saunders, K.O.; Lee, E.; Parks, R.; Martinez, D.R.; Li, D.; Chen, H.; Edwards, R.J.; Gobeil, S.; Barr, M.; Mansouri, K.; et al. Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses. Nature 2021, 594, 553–559. [Google Scholar] [CrossRef]
  91. Dickey, T.H.; Ma, R.; Orr-Gonzalez, S.; Ouahes, T.; Patel, P.; McAleese, H.; Butler, B.; Eudy, E.; Eaton, B.; Murphy, M.; et al. Design of a stabilized RBD enables potently neutralizing SARS-CoV-2 single-component nanoparticle vaccines. Cell Rep 2023, 42, 112266. [Google Scholar] [CrossRef] [PubMed]
  92. Ladenstein, R.; Morgunova, E. Second career of a biosynthetic enzyme: Lumazine synthase as a virus-like nanoparticle in vaccine development. Biotechnol Rep (Amst) 2020, 27, e00494. [Google Scholar] [CrossRef] [PubMed]
  93. Ladenstein, R.; Schneider, M.; Huber, R.; Bartunik, H.D.; Wilson, K.; Schott, K.; Bacher, A. Heavy riboflavin synthase from Bacillus subtilis. Crystal structure analysis of the icosahedral beta 60 capsid at 3.3 A resolution. J Mol Biol 1988, 203, 1045–1070. [Google Scholar] [CrossRef] [PubMed]
  94. Zhang, X.; Meining, W.; Fischer, M.; Bacher, A.; Ladenstein, R. X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile Aquifex aeolicus at 1.6 A resolution: determinants of thermostability revealed from structural comparisons. J Mol Biol 2001, 306, 1099–1114. [Google Scholar] [CrossRef] [PubMed]
  95. Zhang, X.; Konarev, P.V.; Petoukhov, M.V.; Svergun, D.I.; Xing, L.; Cheng, R.H.; Haase, I.; Fischer, M.; Bacher, A.; Ladenstein, R.; et al. Multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly. J Mol Biol 2006, 362, 753–770. [Google Scholar] [CrossRef] [PubMed]
  96. Jardine, J.; Julien, J.P.; Menis, S.; Ota, T.; Kalyuzhniy, O.; McGuire, A.; Sok, D.; Huang, P.S.; MacPherson, S.; Jones, M.; et al. Rational HIV immunogen design to target specific germline B cell receptors. Science 2013, 340, 711–716. [Google Scholar] [CrossRef]
  97. Jardine, J.G.; Ota, T.; Sok, D.; Pauthner, M.; Kulp, D.W.; Kalyuzhniy, O.; Skog, P.D.; Thinnes, T.C.; Bhullar, D.; Briney, B.; et al. HIV-1 VACCINES. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science 2015, 349, 156–161. [Google Scholar] [CrossRef] [PubMed]
  98. Geng, Q.; Tai, W.; Baxter, V.K.; Shi, J.; Wan, Y.; Zhang, X.; Montgomery, S.A.; Taft-Benz, S.A.; Anderson, E.J.; Knight, A.C.; et al. Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection. PLoS Pathog 2021, 17, e1009897. [Google Scholar] [CrossRef] [PubMed]
  99. Argentinian AntiCovid, C. Covalent coupling of Spike’s receptor binding domain to a multimeric carrier produces a high immune response against SARS-CoV-2. Sci Rep 2022, 12, 692. [Google Scholar] [CrossRef]
  100. Lu, Y.; Li, Q.; Fan, H.; Liao, C.; Zhang, J.; Hu, H.; Yi, H.; Peng, Y.; Lu, J.; Chen, Z. A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529). Int J Nanomedicine 2023, 18, 353–367. [Google Scholar] [CrossRef]
  101. Okba, N.M.A.; Widjaja, I.; van Dieren, B.; Aebischer, A.; van Amerongen, G.; de Waal, L.; Stittelaar, K.J.; Schipper, D.; Martina, B.; van den Brand, J.M.A.; et al. Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV. Emerg Microbes Infect 2020, 9, 1080–1091. [Google Scholar] [CrossRef]
  102. Malonis, R.J.; Georgiev, G.I.; Haslwanter, D.; VanBlargan, L.A.; Fallon, G.; Vergnolle, O.; Cahill, S.M.; Harris, R.; Cowburn, D.; Chandran, K.; et al. A Powassan virus domain III nanoparticle immunogen elicits neutralizing and protective antibodies in mice. PLoS Pathog 2022, 18, e1010573. [Google Scholar] [CrossRef]
  103. Ren, X.; Cao, N.; Tian, L.; Liu, W.; Zhu, H.; Rong, Z.; Yao, M.; Li, X.; Qian, P. A self-assembled nanoparticle vaccine based on pseudorabies virus glycoprotein D induces potent protective immunity against pseudorabies virus infection. Vet Microbiol 2023, 284, 109799. [Google Scholar] [CrossRef]
  104. Zhu, H.; Li, X.; Ren, X.; Chen, H.; Qian, P. Improving cross-protection against influenza virus in mice using a nanoparticle vaccine of mini-HA. Vaccine 2022, 40, 6352–6361. [Google Scholar] [CrossRef]
  105. Alvarez, P.; Zylberman, V.; Ghersi, G.; Boado, L.; Palacios, C.; Goldbaum, F.; Mattion, N. Tandem repeats of the extracellular domain of Matrix 2 influenza protein exposed in Brucella lumazine synthase decameric carrier molecule induce protection in mice. Vaccine 2013, 31, 806–812. [Google Scholar] [CrossRef] [PubMed]
  106. Kang, Y.F.; Zhang, X.; Yu, X.H.; Zheng, Q.; Liu, Z.; Li, J.P.; Sun, C.; Kong, X.W.; Zhu, Q.Y.; Chen, H.W.; et al. Immunization with a Self-Assembled Nanoparticle Vaccine Elicits Potent Neutralizing Antibody Responses against EBV Infection. Nano Lett 2021, 21, 2476–2486. [Google Scholar] [CrossRef] [PubMed]
  107. Wichgers Schreur, P.J.; Tacken, M.; Gutjahr, B.; Keller, M.; van Keulen, L.; Kant, J.; van de Water, S.; Lin, Y.; Eiden, M.; Rissmann, M.; et al. Vaccine Efficacy of Self-Assembled Multimeric Protein Scaffold Particles Displaying the Glycoprotein Gn Head Domain of Rift Valley Fever Virus. Vaccines (Basel) 2021, 9. [Google Scholar] [CrossRef] [PubMed]
  108. McHugh, C.A.; Fontana, J.; Nemecek, D.; Cheng, N.; Aksyuk, A.A.; Heymann, J.B.; Winkler, D.C.; Lam, A.S.; Wall, J.S.; Steven, A.C.; et al. A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. EMBO J 2014, 33, 1896–1911. [Google Scholar] [CrossRef]
  109. Sutter, M.; Boehringer, D.; Gutmann, S.; Gunther, S.; Prangishvili, D.; Loessner, M.J.; Stetter, K.O.; Weber-Ban, E.; Ban, N. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol 2008, 15, 939–947. [Google Scholar] [CrossRef]
  110. Akita, F.; Chong, K.T.; Tanaka, H.; Yamashita, E.; Miyazaki, N.; Nakaishi, Y.; Suzuki, M.; Namba, K.; Ono, Y.; Tsukihara, T.; et al. The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses. J Mol Biol 2007, 368, 1469–1483. [Google Scholar] [CrossRef]
  111. Lagoutte, P.; Mignon, C.; Stadthagen, G.; Potisopon, S.; Donnat, S.; Mast, J.; Lugari, A.; Werle, B. Simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design. Vaccine 2018, 36, 3622–3628. [Google Scholar] [CrossRef] [PubMed]
  112. Kar, U.; Khaleeq, S.; Garg, P.; Bhat, M.; Reddy, P.; Vignesh, V.S.; Upadhyaya, A.; Das, M.; Chakshusmathi, G.; Pandey, S.; et al. Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens. Front Immunol 2022, 13, 890622. [Google Scholar] [CrossRef]
  113. Khaleeq, S.; Sengupta, N.; Kumar, S.; Patel, U.R.; Rajmani, R.S.; Reddy, P.; Pandey, S.; Singh, R.; Dutta, S.; Ringe, R.P.; et al. Neutralizing Efficacy of Encapsulin Nanoparticles against SARS-CoV2 Variants of Concern. Viruses 2023, 15. [Google Scholar] [CrossRef] [PubMed]
  114. Corrigan, A.R.; Duan, H.; Cheng, C.; Gonelli, C.A.; Ou, L.; Xu, K.; DeMouth, M.E.; Geng, H.; Narpala, S.; O’Connell, S.; et al. Fusion peptide priming reduces immune responses to HIV-1 envelope trimer base. Cell Rep 2021, 35, 108937. [Google Scholar] [CrossRef] [PubMed]
  115. Wang, Z.; Zhang, B.; Ou, L.; Qiu, Q.; Wang, L.; Bylund, T.; Kong, W.P.; Shi, W.; Tsybovsky, Y.; Wu, L.; et al. Extraordinary Titer and Broad Anti-SARS-CoV-2 Neutralization Induced by Stabilized RBD Nanoparticles from Strain BA.5. Vaccines (Basel) 2023, 12. [Google Scholar] [CrossRef] [PubMed]
  116. Henderson, C.E.; Perham, R.N.; Finch, J.T. Structure and symmetry of B. stearothermophilus pyruvate dehydrogenase multienzyme complex and implications for eucaryote evolution. Cell 1979, 17, 85–93. [Google Scholar] [CrossRef]
  117. Milne, J.L.; Wu, X.; Borgnia, M.J.; Lengyel, J.S.; Brooks, B.R.; Shi, D.; Perham, R.N.; Subramaniam, S. Molecular structure of a 9-MDa icosahedral pyruvate dehydrogenase subcomplex containing the E2 and E3 enzymes using cryoelectron microscopy. J Biol Chem 2006, 281, 4364–4370. [Google Scholar] [CrossRef] [PubMed]
  118. Caivano, A.; Doria-Rose, N.A.; Buelow, B.; Sartorius, R.; Trovato, M.; D’Apice, L.; Domingo, G.J.; Sutton, W.F.; Haigwood, N.L.; De Berardinis, P. HIV-1 Gag p17 presented as virus-like particles on the E2 scaffold from Geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of IFNgamma production by CD4+ T cells. Virology 2010, 407, 296–305. [Google Scholar] [CrossRef]
  119. De Berardinis, P.; Sartorius, R.; Caivano, A.; Mascolo, D.; Domingo, G.J.; Del Pozzo, G.; Gaubin, M.; Perham, R.N.; Piatier-Tonneau, D.; Guardiola, J. Use of fusion proteins and procaryotic display systems for delivery of HIV-1 antigens: development of novel vaccines for HIV-1 infection. Curr HIV Res 2003, 1, 441–446. [Google Scholar] [CrossRef]
  120. Domingo, G.J.; Caivano, A.; Sartorius, R.; Barba, P.; Backstrom, M.; Piatier-Tonneau, D.; Guardiola, J.; De Berardinis, P.; Perham, R.N. Induction of specific T-helper and cytolytic responses to epitopes displayed on a virus-like protein scaffold derived from the pyruvate dehydrogenase multienzyme complex. Vaccine 2003, 21, 1502–1509. [Google Scholar] [CrossRef]
  121. Jaworski, J.P.; Krebs, S.J.; Trovato, M.; Kovarik, D.N.; Brower, Z.; Sutton, W.F.; Waagmeester, G.; Sartorius, R.; D’Apice, L.; Caivano, A.; et al. Co-immunization with multimeric scaffolds and DNA rapidly induces potent autologous HIV-1 neutralizing antibodies and CD8+ T cells. PLoS One 2012, 7, e31464. [Google Scholar] [CrossRef] [PubMed]
  122. He, L.; Chaudhary, A.; Lin, X.; Sou, C.; Alkutkar, T.; Kumar, S.; Ngo, T.; Kosviner, E.; Ozorowski, G.; Stanfield, R.L.; et al. Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines. Nat Commun 2021, 12, 2633. [Google Scholar] [CrossRef] [PubMed]
  123. Kim, K.K.; Kim, R.; Kim, S.H. Crystal structure of a small heat-shock protein. Nature 1998, 394, 595–599. [Google Scholar] [CrossRef] [PubMed]
  124. Wiley, J.A.; Richert, L.E.; Swain, S.D.; Harmsen, A.; Barnard, D.L.; Randall, T.D.; Jutila, M.; Douglas, T.; Broomell, C.; Young, M.; et al. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses. PLoS One 2009, 4, e7142. [Google Scholar] [CrossRef] [PubMed]
  125. Flenniken, M.L.; Willits, D.A.; Brumfield, S.; Young, M.J.; Douglas, T. The Small Heat Shock Protein Cage from Methanococcus jannaschii Is a Versatile Nanoscale Platform for Genetic and Chemical Modification. Nano Letters 2003, 3, 1573–1576. [Google Scholar] [CrossRef]
  126. Crowther, R.A.; Kiselev, N.A.; Bottcher, B.; Berriman, J.A.; Borisova, G.P.; Ose, V.; Pumpens, P. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell 1994, 77, 943–950. [Google Scholar] [CrossRef] [PubMed]
  127. Ulrich, R.; Nassal, M.; Meisel, H.; Krüger, D.H. Core particles of hepatitis B virus as carrier for foreign epitopes. Adv Virus Res 1998, 50, 141–182. [Google Scholar] [CrossRef] [PubMed]
  128. Peyret, H.; Gehin, A.; Thuenemann, E.C.; Blond, D.; El Turabi, A.; Beales, L.; Clarke, D.; Gilbert, R.J.C.; Fry, E.E.; Stuart, D.I.; et al. Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins. PLOS ONE 2015, 10, e0120751. [Google Scholar] [CrossRef]
  129. Lee, B.O.; Tucker, A.; Frelin, L.; Sallberg, M.; Jones, J.; Peters, C.; Hughes, J.; Whitacre, D.; Darsow, B.; Peterson, D.L.; et al. Interaction of the hepatitis B core antigen and the innate immune system. J Immunol 2009, 182, 6670–6681. [Google Scholar] [CrossRef]
  130. Cheng, R.H.; Olson, N.H.; Baker, T.S. Cauliflower mosaic virus: a 420 subunit (T = 7), multilayer structure. Virology 1992, 186, 655–668. [Google Scholar] [CrossRef]
  131. Zeltins, A.; West, J.; Zabel, F.; El Turabi, A.; Balke, I.; Haas, S.; Maudrich, M.; Storni, F.; Engeroff, P.; Jennings, G.T.; et al. Incorporation of tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, Alzheimer’s and cat allergy. NPJ Vaccines 2017, 2, 30. [Google Scholar] [CrossRef] [PubMed]
  132. Cabral-Miranda, G.; Lim, S.M.; Mohsen, M.O.; Pobelov, I.V.; Roesti, E.S.; Heath, M.D.; Skinner, M.A.; Kramer, M.F.; Martina, B.E.E.; Bachmann, M.F. Zika Virus-Derived E-DIII Protein Displayed on Immunologically Optimized VLPs Induces Neutralizing Antibodies without Causing Enhancement of Dengue Virus Infection. Vaccines (Basel) 2019, 7. [Google Scholar] [CrossRef] [PubMed]
  133. Chang, X.; Liu, X.; Mohsen, M.O.; Zeltins, A.; Martina, B.; Vogel, M.; Bachmann, M.F. Induction of Broadly Cross-Reactive Antibodies by Displaying Receptor Binding Domains of SARS-CoV-2 on Virus-like Particles. Vaccines (Basel) 2022, 10. [Google Scholar] [CrossRef] [PubMed]
  134. Zha, L.; Chang, X.; Zhao, H.; Mohsen, M.O.; Hong, L.; Zhou, Y.; Chen, H.; Liu, X.; Zhang, J.; Li, D.; et al. Development of a Vaccine against SARS-CoV-2 Based on the Receptor-Binding Domain Displayed on Virus-Like Particles. Vaccines (Basel) 2021, 9. [Google Scholar] [CrossRef] [PubMed]
  135. Chang, X.; Zeltins, A.; Mohsen, M.O.; Gharailoo, Z.; Zha, L.; Liu, X.; Walton, S.; Vogel, M.; Bachmann, M.F. A Novel Double Mosaic Virus-like Particle-Based Vaccine against SARS-CoV-2 Incorporates Both Receptor Binding Motif (RBM) and Fusion Domain. Vaccines (Basel) 2021, 9. [Google Scholar] [CrossRef] [PubMed]
  136. Mohsen, M.O.; Balke, I.; Zinkhan, S.; Zeltina, V.; Liu, X.; Chang, X.; Krenger, P.S.; Plattner, K.; Gharailoo, Z.; Vogt, A.S.; et al. A scalable and highly immunogenic virus-like particle-based vaccine against SARS-CoV-2. Allergy 2022, 77, 243–257. [Google Scholar] [CrossRef] [PubMed]
  137. Golmohammadi, R.; Fridborg, K.; Bundule, M.; Valegård, K.; Liljas, L. The crystal structure of bacteriophage Qβ at 3.5 å resolution. Structure 1996, 4, 543–554. [Google Scholar] [CrossRef]
  138. Bessa, J.; Schmitz, N.; Hinton, H.J.; Schwarz, K.; Jegerlehner, A.; Bachmann, M.F. Efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design. Eur J Immunol 2008, 38, 114–126. [Google Scholar] [CrossRef] [PubMed]
  139. van den Worm, S.; Koning, R.; Warmenhoven, H.; Koerten, H.K.; Duin, J. Cryo Electron Microscopy Reconstructions of the Leviviridae Unveil the Densest Icosahedral RNA Packing Possible. Journal of molecular biology 2006, 363, 858–865. [Google Scholar] [CrossRef]
  140. Shishovs, M.; Rumnieks, J.; Diebolder, C.; Jaudzems, K.; Andreas, L.B.; Stanek, J.; Kazaks, A.; Kotelovica, S.; Akopjana, I.; Pintacuda, G.; et al. Structure of AP205 Coat Protein Reveals Circular Permutation in ssRNA Bacteriophages. J Mol Biol 2016, 428, 4267–4279. [Google Scholar] [CrossRef]
  141. Escolano, A.; Gristick, H.B.; Abernathy, M.E.; Merkenschlager, J.; Gautam, R.; Oliveira, T.Y.; Pai, J.; West, A.P., Jr.; Barnes, C.O.; Cohen, A.A.; et al. Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques. Nature 2019, 570, 468–473. [Google Scholar] [CrossRef] [PubMed]
  142. Cohen, A.A.; Yang, Z.; Gnanapragasam, P.N.P.; Ou, S.; Dam, K.A.; Wang, H.; Bjorkman, P.J. Construction, characterization, and immunization of nanoparticles that display a diverse array of influenza HA trimers. PLoS One 2021, 16, e0247963. [Google Scholar] [CrossRef] [PubMed]
  143. Liu, X.; Chang, X.; Rothen, D.; Derveni, M.; Krenger, P.; Roongta, S.; Wright, E.; Vogel, M.; Tars, K.; Mohsen, M.O.; et al. AP205 VLPs Based on Dimerized Capsid Proteins Accommodate RBM Domain of SARS-CoV-2 and Serve as an Attractive Vaccine Candidate. Vaccines (Basel) 2021, 9. [Google Scholar] [CrossRef] [PubMed]
  144. Fougeroux, C.; Goksoyr, L.; Idorn, M.; Soroka, V.; Myeni, S.K.; Dagil, R.; Janitzek, C.M.; Sogaard, M.; Aves, K.L.; Horsted, E.W.; et al. Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity. Nat Commun 2021, 12, 324. [Google Scholar] [CrossRef] [PubMed]
  145. Peabody, D.S.; Manifold-Wheeler, B.; Medford, A.; Jordan, S.K.; do Carmo Caldeira, J.; Chackerian, B. Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2. J Mol Biol 2008, 380, 252–263. [Google Scholar] [CrossRef]
  146. Chiba, S.; Frey, S.J.; Halfmann, P.J.; Kuroda, M.; Maemura, T.; Yang, J.E.; Wright, E.R.; Kawaoka, Y.; Kane, R.S. Multivalent nanoparticle-based vaccines protect hamsters against SARS-CoV-2 after a single immunization. Communications Biology 2021, 4, 597. [Google Scholar] [CrossRef] [PubMed]
  147. DiMaio, F.; Leaver-Fay, A.; Bradley, P.; Baker, D.; Andre, I. Modeling symmetric macromolecular structures in Rosetta3. PLoS One 2011, 6, e20450. [Google Scholar] [CrossRef] [PubMed]
  148. Hsia, Y.; Bale, J.B.; Gonen, S.; Shi, D.; Sheffler, W.; Fong, K.K.; Nattermann, U.; Xu, C.; Huang, P.S.; Ravichandran, R.; et al. Design of a hypersTable 60-subunit protein dodecahedron. [corrected]. Nature 2016, 535, 136–139. [Google Scholar] [CrossRef]
  149. Bale, J.B.; Gonen, S.; Liu, Y.; Sheffler, W.; Ellis, D.; Thomas, C.; Cascio, D.; Yeates, T.O.; Gonen, T.; King, N.P.; et al. Accurate design of megadalton-scale two-component icosahedral protein complexes. Science 2016, 353, 389–394. [Google Scholar] [CrossRef]
  150. Smith, G.; Raghunandan, R.; Wu, Y.; Liu, Y.; Massare, M.; Nathan, M.; Zhou, B.; Lu, H.; Boddapati, S.; Li, J.; et al. Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats. PLoS One 2012, 7, e50852. [Google Scholar] [CrossRef]
  151. Swanson, K.A.; Settembre, E.C.; Shaw, C.A.; Dey, A.K.; Rappuoli, R.; Mandl, C.W.; Dormitzer, P.R.; Carfi, A. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc Natl Acad Sci U S A 2011, 108, 9619–9624. [Google Scholar] [CrossRef] [PubMed]
  152. August, A.; Glenn, G.M.; Kpamegan, E.; Hickman, S.P.; Jani, D.; Lu, H.; Thomas, D.N.; Wen, J.; Piedra, P.A.; Fries, L.F. A Phase 2 randomized, observer-blind, placebo-controlled, dose-ranging trial of aluminum-adjuvanted respiratory syncytial virus F particle vaccine formulations in healthy women of childbearing age. Vaccine 2017, 35, 3749–3759. [Google Scholar] [CrossRef] [PubMed]
  153. Marcandalli, J.; Fiala, B.; Ols, S.; Perotti, M.; de van der Schueren, W.; Snijder, J.; Hodge, E.; Benhaim, M.; Ravichandran, R.; Carter, L.; et al. Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus. Cell 2019, 176, 1420–1431. [Google Scholar] [CrossRef] [PubMed]
  154. Boyoglu-Barnum, S.; Ellis, D.; Gillespie, R.A.; Hutchinson, G.B.; Park, Y.J.; Moin, S.M.; Acton, O.J.; Ravichandran, R.; Murphy, M.; Pettie, D.; et al. Quadrivalent influenza nanoparticle vaccines induce broad protection. Nature 2021, 592, 623–628. [Google Scholar] [CrossRef] [PubMed]
  155. Sanders, R.W.; Derking, R.; Cupo, A.; Julien, J.P.; Yasmeen, A.; de Val, N.; Kim, H.J.; Blattner, C.; de la Pena, A.T.; Korzun, J.; et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog 2013, 9, e1003618. [Google Scholar] [CrossRef] [PubMed]
  156. Brouwer, P.J.M.; Antanasijevic, A.; Berndsen, Z.; Yasmeen, A.; Fiala, B.; Bijl, T.P.L.; Bontjer, I.; Bale, J.B.; Sheffler, W.; Allen, J.D.; et al. Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle. Nat Commun 2019, 10, 4272. [Google Scholar] [CrossRef] [PubMed]
  157. Brouwer, P.J.M.; Antanasijevic, A.; de Gast, M.; Allen, J.D.; Bijl, T.P.L.; Yasmeen, A.; Ravichandran, R.; Burger, J.A.; Ozorowski, G.; Torres, J.L.; et al. Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles. NPJ Vaccines 2021, 6, 24. [Google Scholar] [CrossRef] [PubMed]
  158. Brouwer, P.J.M.; Brinkkemper, M.; Maisonnasse, P.; Dereuddre-Bosquet, N.; Grobben, M.; Claireaux, M.; de Gast, M.; Marlin, R.; Chesnais, V.; Diry, S.; et al. Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection. Cell 2021, 184, 1188–1200.e1119. [Google Scholar] [CrossRef]
  159. Kang, Y.F.; Sun, C.; Sun, J.; Xie, C.; Zhuang, Z.; Xu, H.Q.; Liu, Z.; Liu, Y.H.; Peng, S.; Yuan, R.Y.; et al. Quadrivalent mosaic HexaPro-bearing nanoparticle vaccine protects against infection of SARS-CoV-2 variants. Nat Commun 2022, 13, 2674. [Google Scholar] [CrossRef]
  160. Arunachalam, P.S.; Walls, A.C.; Golden, N.; Atyeo, C.; Fischinger, S.; Li, C.; Aye, P.; Navarro, M.J.; Lai, L.; Edara, V.V.; et al. Adjuvanting a subunit COVID-19 vaccine to induce protective immunity. Nature 2021, 594, 253–258. [Google Scholar] [CrossRef]
  161. Walls, A.C.; Miranda, M.C.; Schafer, A.; Pham, M.N.; Greaney, A.; Arunachalam, P.S.; Navarro, M.J.; Tortorici, M.A.; Rogers, K.; O’Connor, M.A.; et al. Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines. Cell 2021, 184, 5432–5447.e5416. [Google Scholar] [CrossRef] [PubMed]
Preprints 105878 g001
Figure 1. Structures of some widely used protein nanoparticle. A. Naturally occurring PNPs. Surface structures of ferritin (PDB: 3BVE), lumazine synthase (PDB: 1HQK), and encapsulin (PDB: 3DKT). B. Capsid/Coat PNPs of viruses and bacteriophages. Surface structures of hepatitis B core antigen, HBcAg(PDB: IQGT), AP205 (PDB: 5LQP), and MS2 (PDB: 2MS2). C. Computationally designed NP. Surface structure of I53-50 (PDB: 6P6F). Ferritin, LS, HBcAg, and MS2 were generated using Chimera 1.17.3 while encapsulin, AP205, and I53-50 were retrieved from Protein Data Bank.
Figure 1. Structures of some widely used protein nanoparticle. A. Naturally occurring PNPs. Surface structures of ferritin (PDB: 3BVE), lumazine synthase (PDB: 1HQK), and encapsulin (PDB: 3DKT). B. Capsid/Coat PNPs of viruses and bacteriophages. Surface structures of hepatitis B core antigen, HBcAg(PDB: IQGT), AP205 (PDB: 5LQP), and MS2 (PDB: 2MS2). C. Computationally designed NP. Surface structure of I53-50 (PDB: 6P6F). Ferritin, LS, HBcAg, and MS2 were generated using Chimera 1.17.3 while encapsulin, AP205, and I53-50 were retrieved from Protein Data Bank.
Preprints 105878 g001
Table 1. Physical characteristics of some PNPs used in vaccine platforms.
Table 1. Physical characteristics of some PNPs used in vaccine platforms.
Nanoparticle Microorganism Triangulation # (Protomers) Size in nm Reference
Ferritin H. pylori 24 12 [49,51]
Lumazine synthase Aquifex aeolicus T=1 (60) 15.4 [93,94]
Bacillus subtilis T=3 (180) 29 [95]
Encapsulin Thermotoga maritima T=1 (60) 24 [109]
Pyrococcus furiosus T=3 (180) 31 [110]
Quasibacillus thermotolerance T=4 (240) 42 [109]
E2p Geobacillus stearothermophilus T=1 (60) 27 [116,117]
sHSP Methanococcus jannaschii 24 12 [123]
HBcAg Hepatitis B virus T=3/T=4 (180/240) 28-32 [126]
CuMVTT Cauliflower mosaic virus T = 7 (420) 53.8 [130]
Qβ Bacteriophage Qβ T = 3 (180) 25 [137]
AP205 coat protein Bacteriophage AP205 T=3 (180) 25-30 [139]
MS2 Bacteriophage MS2 T=3 (180) 27 [145]
I53-50 Synthetic T=2 (120) 30 [149]
Table 2. Protein based nanoparticles and their use in vaccine development.
Table 2. Protein based nanoparticles and their use in vaccine development.
Nanoparticle Pathogen Antigen References
Ferritin Influenza Hemagglutinin (HA)/
Ectodomain of HA/
HA stem domain		 [55,56,58,59]
HIV Envelope glycoprotein trimers/
ConM SOSIP trimer/
Native-like BG505 SOSIP trimers		 [60,63,64,65]
HCV sE2 protein [68]
RSV pre-fusion Fusion (F) [69]
ZIKV E protein domain III (EDIII) [70,71]
EBV RBD of gp350/gH/gL or both gH/gL and gp42 glycoproteins [72,73]
SARS-CoV-2 Full-length spike (S) protein/ S ectodomain with deletion of 70 C-terminal residues/ RBD/RBD and heptad repeat (HR)/ Non-glycosylated RBD immunogen. [77,79,80,82,83,88,90,91]
Lumazine synthase HIV Surface glycoprotein (gp120) [60,96]
SARS CoV-2 S protein trimer/ RBD/ RBD-specific nanobodies obtained from a naïve alpaca phage display library [78,91,98,99,100]
MERS-CoV Multimeric RBD [101]
Powassan virus EDIII [102]
Pseudorabies virus glycoprotein D [103]
Influenza Ectodomain of the Matrix 2 (M2e)/mini-HA [104,105]
EBV gp350 [106]
Rift Valley fever virus Head domain (Gn) [107]
Encapsulin Influenza M2e/HA [111,112]
EBV The domains I, II, and III of gp350 [72]
SARS CoV-2 RBD [113]
Encapsulin (EnDS) SARS CoV-2 WA1 and BA.5 RBD [115]
E2p HIV B and T cell epitopes of HIV-1/ gp140 [60,118,121]
Ebola virus GP [122]
SARS-CoV-2 RBD [91]
HBcAg ZIKV EDIII [129]
CuMVtt ZIKV EDIII [132]
SARS-CoV-2 RBD/RBM/fusion peptide [133,134,135,136]
Qβ VLPs Influenza M2e [138]
AP205 HIV HIV Envelope protein [141]
Influenza HA (mosaic) [142]
SARS CoV-2 RBD [143,144]
MS2 SARS-CoV-2 S protein [146]
I53-50 RSV F protein/trimeric DS-Cav1 [150,151,152,153]
Quadrivalent influenza vaccine candidate HA trimers [154]
HIV SOSIP trimers [156]
SARS CoV-2 Full-length S/Mosaic S /RBD/Mosaic RBD from multiple sarbecoviruses [76,158,159,160,161]
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