The 3Ds in virus‐like particle based‐vaccines: “Design, Delivery and Dynamics”

Abstract Vaccines need to be rationally designed in order be delivered to the immune system for maximizing induction of dynamic immune responses. Virus‐like particles (VLPs) are ideal platforms for such 3D vaccines, as they allow the display of complex and native antigens in a highly repetitive form on their surface and can easily reach lymphoid organs in intact form for optimal activation of B and T cells. Adjusting size and zeta potential may allow investigators to further fine‐tune delivery to lymphoid organs. An additional way to alter vaccine transfer to lymph nodes and spleen may be the formulation with micron‐sized adjuvants that creates a local depot and results in a slow release of antigen and adjuvant. Ideally, the adjuvant in addition stimulates the innate immune system. The dynamics of the immune response may be further enhanced by inclusion of Toll‐like receptor ligands, which many VLPs naturally package. Hence, considering the 3Ds in vaccine development may allow for enhancement of their attributes to tackle complex diseases, not usually amenable to conventional vaccine strategies.


| B RIEF LOOK AT THE HIS TORY OF VACCINE S: WHERE WE ARE COMING FROM
Viruses are highly evolved and adapted supramolecular entities that take advantage of their host and can be strongly pathogenic. 1 Viruses can cause pathogenesis by their entry and replication in host cells. Damage is usually caused by lytic activity of the virus or killing infected cells mediated by the immune cells, resulting in excessive cell death and inflammation. Hence, early stop of viral spread is the goal of most vaccinations. 2 The practice of variolation, also called "inoculation or insertion," was used in China in the 10th century to immunize people against smallpox. Live, virulent viruses were collected from scabs of infected victims and inserted into the skin of healthy individuals.
The concept of variolation against smallpox spread further to India in the 17th century followed to southwest Asia and Balkans 3,4 and even to London by Lady Mary Montagu. 5 Variolation was considered the sole practice to induce protection against smallpox until 1796 when Edward Jenner tested his hypothesis that pus from blisters from cowpox-infected milkmaids can protect against smallpox. Jenner inoculated an 8-year-old boy, James Phipps, in both arms with this cow-derived virus, causing the boy to develop some mild symptoms. Later, he challenged the boy with smallpox (by way of variolation), and there were no signs of disease. 6,7 The method was termed vaccination from the Latin word vacca, which means cow, and was much safer with milder reactions than variolation (which caused up to 1% death) and no risk of disease transmission. 8 Mass vaccination against smallpox, finally pioneered by the World techniques not yet developed. Nevertheless, Pasteur could successfully protect dogs from rabies as well as a 9-year-old boy who had been severely attacked by infected dogs with a vaccine that was, however, produced in the brain of infected rabbits. 9 In the late 19th century, a vaccine against plague (Yersinia pestis) was started by Alexandre Yersin based on killed whole-cell bacteria. However, the vaccine proved less efficacious against the most severe pneumonia form of plague. 10 For this reason and due to re-emergence of plague, efforts are currently made to develop more effective vaccines against plague based on attenuated bacteria and recombinant proteins. 11 The development of a vaccine against tuberculosis occurred during the period of 1890 to 1950. At the end of the 19th century, Calmette and Guérin started their research for a vaccine against tuberculosis at the Pasteur Institute, resulting in the Bacillus Calmette-Guérin (BCG) vaccine strain. 12 The efficacy of this BCG vaccine is still controversial, despite the fact that live BCG is in use as a vaccine against TB in humans in some parts of the world where it seems to attenuate severe forms of TB. 13,14 The Salk vaccine "inactivated poliovirus" was developed by Jonas Salk in the 1950s, while the Sabin vaccine "live attenuated poliovirus" was advanced by Albert Sabin for oral administration. Though highly efficacious, the Sabin vaccine is only used rarely nowadays due to the potential to revert to a virulent form or cause disease in immunocompromised individuals. Mass immunization against poliovirus resulted in the eradication of the disease in most parts of the world. 15 The widely used "MMR" vaccines against "measles, mumps, and rubella" have been developed using attenuated viruses. 16 The MMR vaccine has massively reduced the presence of these viruses in our society and, in terms of safety and, portrays a positive benefit-risk profile. 17 Vaccines may not only be used to protect against different pathogens, but they also play an important role in the treatment of allergies. 18 Furthermore, and perhaps with the brightest future, a large number of therapeutic vaccines are currently developed for the treatment of cancer and other non-communicable diseases, such as hypertension, psoriasis, asthma, and type II diabetes.
Taken together, traditional vaccines (inactivated or attenuated pathogens or toxins) have shown high immunogenicity and ability to stimulate the innate and adaptive immune system conferring longterm memory and protection. Such high immunogenicity, particularly where viral vaccines are concerned, is due to several factors such as the ability of the pathogen to replicate, the repetitive surface geometry of the virus and its particulate nature. 19 Yet, several drawbacks in the traditional vaccines have been raised by policymakers, including, but not limited to, safety issues from using the whole pathogen and the difficulty in mass production.
The 21st century has witnessed an advancement in molecular genetics, microbiology, immunology, and genomics techniques in applied vaccinology. Scientists have taken advantage of viruses by making them invaluable sources for generating safe and novel nanoscale scaffolds termed virus-like particles (VLPs). VLPs are multiprotein structures that resemble viruses in many characteristics. 20 The most important difference is that VLPs lack any genetic material and therefore cannot replicate in host cells. 21

| VLP geometry
Crick and Watson have stated in 1956 that "it is a striking fact that almost all small viruses are rods or spheres." "These shells are constructed from a large number of identical protein molecules, of small or moderate size, packed together in a regular manner". 22 A key reason for such an arrangement is the small genomes of many viruses, in particular RNA viruses, which have no space to encode multiple proteins to build up a complex surface. As repetitiveness is unavoidable, the quasicrystalline nature of VLPs serves as a potent pathogen-associated structural pattern (PASP) recognized by both innate and adaptive immune systems. 23 The majority of viruses have a coat or a nucleocapsid consisting of multiple copies of single or just a few different kinds of structural proteins arranged in icosahedral or helical shapes. Coat proteins (CPs) can self-assemble in artificial expression systems or in vitro spontaneously with or without the aid of nucleic acids (NA) or scaffold proteins. According to purely mathematical principles, an icosahedron is built from 60 identical units arranged in 5-3-2 symmetry which is used to describe the rotation possibilities of an icosahedron around an axis. 24,25 Indeed, Crick and Watson predicted that icosahedral viruses must have at least 60 subunits. 22 It has also been shown that an icosahedron with certain multiples of 60 subunits can be formed by incorporating subunits arranged in pentamers and hexamers. 26 However, in this case all subunits cannot be structurally identical as different environments (3D structures) exist within pentamers and hexamers. In practice, often a single CP species can adopt minor structural changes required for pentameric or hexameric contacts, a concept called quasi-equivalence. The number and arrangement of the different subunits of the virus coat can be classified using the quasi-equivalence theory described by Casper and Klug in 1962 based on triangulation. 27 The triangulation number (T) of the coat is determined by two integers h and k and is defined as T = h 2 +hk + k 2 . 27 Essentially, h and k correspond to the number of steps, which must be traveled along two axes in a hexagonal grid across the centers of pentameric and hexameric subunits in order to reach one vertex of icosahedron from another. Figure 1 illustrates icosahedral VLPs with T = 1 coat symmetry using porcine circovirus serotype 2 (PCV2) ( Figure 1A) and T = 3 or T = 4 coat symmetry using HBV core (HBc) ( Figure 1B,C). Hepatitis B virus has an unusual feature that it can be found in both T = 3 or T = 4 forms, 28 albeit the infectious virions appear to be T = 4 only. 29,30 It is interesting to note that most artificially designed synthetic VLPs consisting of a trimerizing and pentamerizing alpha-helical domain form T = 1 particle with 60 spikes. Due to several reasons listed in Table 1, small synthetic T = 1 VLPs may often be not ideal for a vaccine platform and T = 3 particles may be more favorable. In general, icosahedron architecture is more prevalent than the helical shape. The definition of helical symmetry covers a broad area of geometries such as stacked rings, rods, or spring-shaped coils. 38 The helical symmetry of VLPs can be measured using the formula P = μ × ρ where P refers to the pitch of the helix, μ is the number of structural units per turn of the helix, and ρ is the axial rise per subunit. A wellknown example of helical rod-shaped VLPs with hollow central channel is the tobacco mosaic virus-like particles. 39 Interestingly, icosahedral VLP may easily be manipulated to form rod-shaped structures by introducing few mutations. It has been shown previously that the icosahedral Qβ-VLPs would assemble in rod-like particles after mutating five a.a. residues in the FG loop of the CP. 40 Presumably, this happens because of overproduction of hexamers over pentamers which leads to formation of prolate icosahedrons, or rods, rather than regular icosahedrons, as demonstrated also in the case when RNA phage coats of genogroups I and II are mixed together and reassembled in vitro. 41 Tobacco mosaic virus-based VLPs have proven in some studies to be an effective platform for the display of epitopes, successfully eliciting immune response against different target pathogens. 42,43 TA B L E 1 Comparison between T = 1 and T = 3 VLPs The distance between each displayed epitope on the surface of T = 1 VLPs is usually less than 5-10 nm The distance between each displayed epitope on the surface of T = 3 VLPs is approximately 5-10 nm, which is ideal for BCR cross-linking and B cell activation 32 Interior cavity Small cavity Large cavity that allows packaging of large amounts of cargo such as nucleic acids, and Tolllike receptor ligands for optimal activation of B and/or T cells 28,33,34 Abbreviation: B-cell receptor; VLPs, virus-like particles F I G U R E 2 Cucumber mosaic virus (CuMV)-derived virus-like particle fused to universal tetanus toxoid (TT) T-cell epitope. General structural features of CuMV TT . A, Surface representation of viral capsid. Its T = 3 symmetry is formed by pentamers of subunit A (blue) and hexamers of subunits B (dark cyan) and C (light cyan). B, Cross section of the particle as shown in (A). The N-termini of both subunits B and C are colored orange to indicate the points of insertion of the tetanus toxoid epitope. C, The same view as in (A) but with the surface lysine residues liable to conjugation highlighted in red. D, Cartoon representation of a dimer formed by subunits B and C. All images generated from PDB 1F15 Remarkably, the CPs of RNA phages can self-assemble not only into the classical T = 3 particles and the above-mentioned rod-like prolate icosahedrons, but also into T = 1 44 and T = 4 particles, 45,46 demonstrating the high plasticity of the assembly process. Generally, manipulating VLPs' structure allows to study the impact of size on drainage dynamics and magnitude of induced immune responses with one and the same VLP monomer, an avenue of research we are currently following. in hexamers in three-fold symmetry 55 as illustrated in Figure 2A and Video S1.

| Binding and decorating functional molecules or ligands to VLPs
The newly developed platform is thought to enhance the interaction between T H cells and B cells under normal as well as more challenging conditions such as those found in aged patients. This is due to the fact that preexisting immunity to the chosen TT epitope is very broad in humans (and animals) as the peptide binds to essentially all human leukocyte antigen-DR isotype molecules and everybody has been immunized many times against TT. 56,57 The TT epitope has been incorporated by genetic fusion to the CuMV viral envelope protein such that the icosahedral particle maintains its capacity to self-assemble without altering its T = 3 icosahedron geometry and without exposing the peptide on the VLP surface, avoiding interference of TT-specific antibodies ( Figure 2B). This has been achieved by replacing the first 12 N-terminal a.a. with the TT epitope. The resulting CuMV TT -VLP platform has several advantages, including the following: (a) It is adaptable to clinical appli- and highly immunogenic in mice, rabbits, dogs, cats, and horses and therefore is also expected to offer a good safety profile and immunogenicity in humans. 55,58 Using this new CuMV TT platform, we have generated a proof of concept for different preclinical vaccines as listed in Table 2.
The most popular targets for modification on VLPs are accessible lysine residues (Lys) on their surface ( Figure 2C).  38 To link target proteins to VLPs without introducing free Cys residues, the SH group can be introduced in a protected form using the chemical cross-linker SATA (N-succinimidyl S-acetylthioacetate) which then can be exposed after treatment with hydroxylamine for conjugation reactions.
SATA also contains an NHS ester facilitating the formation of an

| VLPs and nucleic acids (NAs)
VLPs can assemble on a polyvalent scaffold of NA, usually RNA.
Generally, the CP of viruses play a role in organizing the packaged NA; conversely, NA plays a role in structure's assembly and immunogenicity of VLPs. A highly ordered NA structure can be seen in bean pod mottle virus, 72 tobacco necrosis virus, 73 or nodaviruses. 74 Bacterial expression systems such as E. coli is the most widely used system for the production of non-enveloped VLPs. VLPs self-assemble on prokaryotic RNA (pRNA) during the expression process. 75 76 The addition of a potent vaccine adjuvant is an essential strategy to activate antigen-presenting cells (APCs) mainly dendritic cells (DCs) for induction of T cell responses. Therefore, the interior surface of VLPs is often exploited to package different TLR ligands such as ssRNA, dsRNA, or CpGs. 77,78 To achieve this, the host NA has to be removed first. This is typically accomplished by the disassembly/reassembly method ( Figure 3A) or by the enzymatic digestion method of the host NA and repackaging with the desired one ( Figure 3B). The second method is the enzymatic digestion method, which is achieved by treating the VLPs with RNase to remove the RNA.

| Routes of administration
The protection conferred by a VLP-based vaccine is essentially dependent on the induced humoral and/or cellular immune response.
However, the science of vaccine administration route is a poorly developed and described area. This is mainly due to the fact that vaccine trials lack standardized comparison of the injection site, needle length, or injection techniques.
Evidence-based medicine aims at improving the quality of health methodologies. Thorough careful assessment of published clinical F I G U R E 3 Virus-like particle (VLP) NA exchange. Two methods for exchanging VLP nucleic acids (NAs). A, Particle disassembly and reassembly. In this method, the particle is initially disassembled, the endogenous NA is removed, and a new NA is repackaged into the VLP during the reassembling process. In the order indicated by the arrows, the images represent the exterior of the particle at the beginning of the process; the interior of the particle packaged with endogenous NA "X"; disassembled particle being reassembled, after removal of NA "X," in the presence of exogenous NA "Y"; the interior of the particle packaged with NA "Y"; and the external view of the particle at the end of the process. B, Enzymatic digestion and infusion. Instead of being washed out after the particle disassembling, in this method the endogenous NA is eliminated by enzymatic digestion. The exogenous NA is then repackaged into the VLP by diffusion, which is facilitated by the high porosity of the particle's surface. In the order indicated by the arrows: the interior of the particle packaged with endogenous NA "X"; digestion of NA "X" and diffusion of exogenous NA "Y" into the VLP; and the interior of the particle packaged with NA "Y." Images generated from unrelated protein data bank (PDB) files 5KIP and 1Y0Q data regarding the route of vaccine administration subcutaneous (SC) vs intramuscular (IM) revealed that the current practice is based on tradition rather than clinical data. 83,84 Traditionally, vaccines were injected SC until it was noticed that adjuvanted vaccines such as Alum could induce unacceptable local reactions at the injection site in humans and injections were changed to IM, most probably because the injection site reactions were less visible. Indeed, the four licensed VLP-based vaccines as well as the first licensed malaria VLP-based vaccine RTS,S (Mosquirix ™ ) use IM route of administration as shown in Table 3.
When comparing SC to IM routes of vaccine injection, IM has shown a faster rate of absorption of administered materials. 85   which induce IgA in a Transforming growth factor beta (TGF-β)-and BLyS-dependent fashion. 104 Hence, systemic IgA responses are similarly regulated as subclass switching to IgG2a as they also require TLR expression in B cells rather than DCs. 105 Recently, it has been shown that administering the influenza VLP vaccine (M2e5x VLP) intranasally facilitated mucosal delivery of the vaccine and can induce a broad cross-protection, prevent weight loss, lower the viral load, attenuate the inflammatory reaction and induce germinal center formation. The study indicated that the observed protective role is managed by B cells as well as CD8 + and CD4 + T cells. 106 In conclusion, it seems that VLPs efficiently reach lymphoid organs from most injection sites and prime similar but not always identical immune responses.

| DYNAMIC S
As discussed above for the delivery, rules for induction of Ab vs T cell responses may differ and both design and delivery of VLPs have important consequences for the dynamics of the immune response.

| VLP-based vaccine formulation
VLP-based vaccines are made of a restricted number of antigens or individual components of the targeted pathogen, hopefully able to confer a protective and/or a therapeutic effect. Accordingly, the protective epitope must be displayed in its native form, at least if the induction of Abs is the desired response. This may be different for T cell-based vaccines, where native display of the epitope is not required. Furthermore, VLP-based vaccines must be optimized for antigen density, dose, and prime/boost interval to obtain a potent Ab response. Unexpectedly, a recent study has demonstrated that such criteria are not necessary for achieving high-avidity T cell responses. 107 Indeed, requirements for vaccines designed to induce Abs (most prophylactic vaccines and vaccines targeting endogenous molecules for their blockage) differ from vaccines aiming to induce T cell responses. In contrast to vaccines against simple viruses, most vaccines targeting complex pathogens will need to induce B and effector T cells. Table 4 summarizes design requirements for optimal delivery to cause a dynamic response of desired specificity.

TA B L E 4
Requirements for optimal induction of Ab or T cell responses or both response, and TLR7 itself may be able to distinguish between prokaryotic and eukaryotic RNA.
Conventionally, vaccines aim to induce long-lived Ab responses rather than memory B cells. 111 We have recently shown that mem-

| T cell responses
A VLP-based vaccine displaying T cell epitopes can elicit a strong T H 1 as well as CTL responses despite the fact that they do not carry any genetic material for replication. [115][116][117][118][119][120] The particulate nature of VLPs allows them to be cross-presented on major histocompatibility class I (MHC-I) molecules as well as on major histocompatibility class II (MHC-II) for effective priming of CD8 + and CD4 + T cells, respectively. 121,122 As mentioned earlier, the type of NAs packaged in the VLPs plays a vital role in determining the desired immune response.
Non-methylated CpGs, TLR9 ligands, are potent stimulators of the innate immune system characterized by upregulating costimulatory molecules, cytokines, and chemokines. 123 Our recent study has investigated the transcriptional signature in DCs from mice vaccinated with Qβ packaging TLR7/8 or TLR9 ligands and displaying H-2D b gp33 epitope. The most striking observation involved CCL2 chemokine which was distinctly expressed in DCs 24 hours following immunization with Qβ(type-B CpGs)-p33, a potent TLR9 ligand.
The identified pathway is thought to play an important role in DCs activation and subsequent induction of potent (CTL) response. 124 Administering synthetic CpGs directly in vivo may result in unfavorable outcomes such as toxic shock, auto-Ab production, inflammation, or the induction of anti-DNA antibodies. 125 Such obstacles can be easily overcome by packaging CpGs in VLPs which indeed reduces side effects and results in an efficient CTL response. 20,81,119 The dogma for an optimal response in VLP-based vaccines is that both antigen and adjuvant should be delivered in the same VLP. 81,126 However, we have shown that this is not always necessary; adjuvants such as CpGs can be packaged in separate VLPs and mixed with the vaccine prior to administration without the need of physical linkage and would still generate a strong CTL response. 33 Similar results were obtained when admixing E7 protein oligomers derived HPV with Qβ-VLPs loaded with CpGs. 127 The formula could induce a protective CD4 + and CD8 + T cell response in a HPV mouse model.
These findings indicate that physical linkage of both antigen and adjuvant in a VLP-based vaccine may not be necessary for effective T cell activation. In contrast, simple, admixing VLPs did not enhance B cell responses, indicating the rules for T and B cell responses are different (see also Table 4).
Adjuvants may enhance the immune response by several mechanisms such as prolonging the release of the antigen at the injection site and upregulating different cytokines, chemokines, and costimulatory molecules. This results in increased maturation and antigen uptake by APCs for effective presentation on MHC-II or by the activation of inflammasomes and TLRs. 128 TLR agonists packaged inside VLPs are recognized by pattern recognition receptors (PRRs) while particulate adjuvants such as Alum are considered damage-associated molecular patterns (DAMPs). As discussed above, adjuvants may also act directly on B cells, as, for example, RNA and CpGs stimulating TLR7/8 and TLR9, respectively.
We have recently harnessed the physiological properties of the lymphatic system by formulating CuMV TT -VLPs displaying H-2D b gp33 peptide derived from lymphocytic choriomeningitis virus with a biodegradable microcrystalline tyrosine depot adjuvant (MCT).
CuMV TT -VLPs are nanoparticles with packaged RNA as TLR7/8 agonist while MCT is a micron-sized depot-forming adjuvant capable of activating the inflammasome. Such formulation has increased the resultant specific CTL response in a murine melanoma model. 62 The novel immune enhancer can also be translated to humans as VLPs used are well defined and the micron-sized adjuvants have been used for decades in allergen-specific desensitization. 129 This micron-/nano-hybrid system harnesses the properties of the lymphatic system optimally, as the micron-sized MCT cannot enter the lymphatics and only the slowly released nanoparticles are actually able to do so, resulting in a slow-release depot of VLPs.

| CON CLUDING REMARK S
The 3Ds of vaccinology, design, delivery, and dynamics, are 3 key components for the efficient generation of a protective vaccine.
Optimal design allows repetitive display of native antigens on VLPs