key: cord-0738556-2m6drxlr
authors: Chung, Young Hun; Cai, Hui; Steinmetz, Nicole F.
title: Viral nanoparticles for drug delivery, imaging, immunotherapy, and theranostic applications
date: 2020-06-27
journal: Adv Drug Deliv Rev
DOI: 10.1016/j.addr.2020.06.024
sha: 1fda76a69438965caf81fe119b71e0c40d2400c5
doc_id: 738556
cord_uid: 2m6drxlr

Viral nanoparticles (VNPs) encompass a diverse array of naturally occurring nanomaterials derived from plant viruses, bacteriophages, and mammalian viruses. The application and development of VNPs and their genome-free versions, the virus-like particles (VLPs), for nanomedicine is a rapidly growing. VLPs can encapsulate a wide range of active ingredients as well as be genetically or chemically conjugated to targeting ligands to achieve tissue specificity. VLPs are manufactured through scalable fermentation or molecular farming, and the materials are biocompatible and biodegradable. These properties have led to a wide range of applications, including cancer therapies, immunotherapies, vaccines, antimicrobial therapies, cardiovascular therapies, gene therapies, as well as imaging and theranostics. The use of VLPs as drug delivery agents is evolving, and sufficient research must continuously be undertaken to translate these therapies to the clinic. This review highlights some of the novel research efforts currently underway in the VNP drug delivery field in achieving this greater goal.

Over the past decades, the progress of nanotechnology has opened up new avenues for medical science, especially in the field of drug delivery. Traditional drug carriers have been developed based on liposomes/lipids [1] , polymers of synthetic and natural origin [2] , and inorganic nanoparticles [3] . To make a clinical impact, nanoparticles for drug delivery must be biocompatible and biodegradable with minimal toxicity. However, some synthetic carriers are restricted by toxicity and/or low delivery efficiency necessitating the development and application of alternate systems [4] . There is no -perfect solution‖, thus there is a need to fuel the development pipeline with novel drug delivery systems. An emerging class of novel drug carriers are protein-based nanoparticles such as protein cages and viruses [5] . Protein cages are self-assembled supramolecular structures arranged nanomedical approaches for drug delivery or imaging aim to utilize biological behaviors to develop smart nanosized cages with high stability, appropriate pharmacokinetics, cell-targeting, and efficient cell penetrability. Plant viruses generally do not exhibit tissue tropisms, therefore these properties can be engineered as an added function to give the nanoparticle formulation, whether of synthetic or natural origin, properties such as cell surface receptor binding, targeting abilities, membrane crossing, and nuclear penetration [9] . : Strategies to carry cargo using VLPs include: a) self-assembly around cargo by altering pH and buffer conditions with CCMV, b) infusion of cargo within RCNMV due to changes in pH and salt concentrations, c) genetic engineering techniques utilizing genetically conjugated scaffolding proteins to encapsulate drugs within P22, d) bioconjugation onto CPMV using exterior surface-exposed residues.

While often depicted as solid nanoparticles (e.g. Figure 1 ), one should keep in mind that many VLPs are porous and the materials -breeze‖ in that they change conformation under various bathing conditions allowing the infusion of small molecules into the capsid's cavity (Figure 2b) . Lommel 

ions from the solvent leading to the formation of 11 to 13 Å-diameter channels; the re-addition of the ions closes the formed channels [20] . Building off of this work, RCNMV was conjugated to a CD46-targeting peptide and loaded with doxorubicin (DOX) through passive diffusion of DOX through the formed channels during the capsid's -open‖ conformation [21] . The targeted and DOX-loaded RCNMV nanoparticles showed significant cytotoxicity in vitro in HeLa cells. Another example is CCMV; changes in pH and salt concentration can affect CCMV capsid stability. Careful adjustment of buffer conditions enables CCMV production in the open and expanded conformation forming pores of ~2 nm allowing for cargo loading [22] . Reversal to the closed and condensed conformation traps the cargo inside in a mechanism referred to as gating [23] .

In addition to passive or non-specific encapsulation, cargo can be tethered and stabilized to the interior of the VLPs making use of bio-specific interactions. For example, Peabody et al. encapsulated cargo into MS2 VLPs by utilizing RNA stem loops fused to the desired cargo. These RNA stem loops are derived from the MS2 genome and bind to the interior of the capsid proteins with high affinity using electrostatic interactions. During infection, these interactions drive assembly and viral genome packing [24] . Additionally, the RNA stem loops can be used to guide cargo to the interior of the MS2 capsids. MS2 particles were packaged with to the interior streptavidin. While the GFP was too large to traverse the viral capsid, the scaffolding proteins could penetrate through thus allowing for exterior functionalization. Genetically enabled loading is particularly powerful, as it improves the quantitative control of the introduced cargos leading to increased homogeneity between samples. The aforementioned methods are not an exhaustive list of all the methods utilized to load cargo into VLPs and other techniques may be applied.

Besides encapsulation techniques, VLPs can also deliver payloads through covalent attachments making use of reactive amino acid side chains of the protein structure. Functional addressable groups are principally amines (Lys), carboxylates (Asp, Glu), thiols (Cys), and aromatic groups (Tyr, Trp), all of which can be utilized for bioconjugation chemistries (Figure 2d ) [28, 29] . Some powerful chemical reactions used for bioconjugation on these moieties include NHS esters, carbodiimides, maleimides, and click chemistries respectively. Moreover, residues such as lysines can be site-specifically engineered into the capsid protein to provide non-native reactive sites [30, 31] .

To increase drug loading capacity, structures of higher order have been introduced.

For instance, layer-by-layer techniques to deliver biomolecules has been widely accomplished with additional compartmentalization even allowing for multipledrug delivery [32] . Hierarchical assembly using virus capsids on DNA origami templates has also been exploited [30] .

Currently, chemotherapy remains the first choice for cancer treatments in the clinic [31] . However, high resistance and recurrence rates, together with rapid drug clearance and non-targeted administration, require the use of maximum-tolerated drug dose in cancer therapy, which may result in high-grade toxicities and limited clinical applicability [33] . Drug delivery systems that can provide targeted and intracellular delivery, promote active drug accumulation in tumor tissues, and minimize required doses would ameliorate these issues and therefore improve therapeutic outcomes.

VLPs possess numerous features that make them highly suitable for targeted delivery of therapeutic agents. In an early study, MS2 VLPs were modified with a targeting peptide (SP94) and were used to selectively deliver chemotherapeutic drugs (DOX, cisplatin and 5-FU) to human hepatocellular carcinoma cells (HCC) [24] . These modified VLPs exhibited high avidity and specificity towards HCC with minimal uptake in healthy cells and induced selective cytotoxicity in vitro J o u r n a l P r e -p r o o f even at very low doses. For instance, the DOX-loaded VLPs killed HCC cells at IC 50 values of 10-15 nM, 20 times better than free DOX.

In fact, various VNP and VLP systems have been utilized for DOX delivery. The loading and release mechanisms of DOX were extensively studied using the aforementioned RCNMV in which the breezing properties of RCNMV were used to encapsulate DOX [34] . Additionally, DOX was electrostatically bound to the exterior of the viral capsid using both phosphate and Tris buffer with the phosphate buffer increasing levels of surface-bound DOX (Figure 3a) . Different [42] . The same is true for other filamentous viral nanoparticles such as PVX. Therefore, from here on out any instances of TMV and PVX will be utilizing the VNP form.

Although not the focus of this review, it is noteworthy to mention that mammalian viruses have also been repurposed to deliver DOX. Some examples include the influenza virus, foot-and-mouth disease virus, the hepatitis B core protein (HBc), and the VLP of Macrobrachium rosenbergii nodavirus [43] [44] [45] [46] .

To increase drug loading potential, polymerization chemistries can be used to increase the functional handles and thus drug loading. An example of this technique has been illustrated using Q formulated as a polymer-protein hybrid nanomaterial [47] . Q was polymerized with an azido-functionalized J o u r n a l P r e -p r o o f oligo(ethylene glycol)-methacrylate (OEGMA-N 3 ) via atom transfer radical polymerization (ATRP), which was then functionalized to DOX through coppercatalyzed azide-alkyne cycloaddition (Figure 3d) . The DOX-Q particles showed comparable cytotoxicity to free DOX in HeLa cells in vitro.

Various VLPs and their incorporation of DOX through infusion, bioconjugation, adsorption, and using polymerization chemistries a) Infusion and surface binding of DOX onto RCNMV VLPs in different buffers to study the binding and release characteristics of DOX from the nanoparticles (reproduced with permission from ref [34] ) b) Bioconjugation of DOX-N-εmaleimidocaproic acid hydrazides onto TMV disk VLPs through maleimide linkages to the cysteine residues (reproduced with permission from ref [36] ) c) Adsorption of DOX onto the exterior surface of PVX (reproduced with permission from ref [40] ) d) Copper-catalyzed azidealkyne cycloaddition of DOX onto OEGMA-N 3 -polymerized Q (reproduced with permission from ref [47] ). Plant virus-based nanoparticles have been widely used for targeted delivery of platinum-based anticancer drugs, which are used in the treatment of nearly 50% of cancer patients undergoing chemotherapy [48] . TMV makes an intriguing platform for efficient delivery of platinum-based drugs such as cisplatin [49] and drug candidate phenanthriplatin (phenPt) [50] as they can be efficiently loaded into the TMV cavity via a charge-driven reaction or by forming stable covalent adducts (Figure 4a,b) . TMV-cisplatin VNP complexes were efficiently taken up by cancer cells and exhibited superior cytotoxicity compared to free cisplatin [49] . For targeted drug delivery, cisplatin-loaded TMV VNPs were modified with mannose and lactose moieties on the exterior surface of the TMV [51] . Through specific recognition between mannose and galectin and lactose and the asialoglycoprotein receptor on the cell membranes, the targeted and drug-loaded TMV showed increased cytotoxicity in MCF-7 and HepG2 cancer cell lines. In a more recent study by the same group, the coat protein of TMV was modified with a small molecular fluorous ponytail at specific sites precipitating self-assembly into spherical nanoparticles (Figure 4c ) [52] . Cisplatin was loaded through metalligand coordination into this spherical assembly with high stability. Graphical illustration of the phenPt docking onto the Glu97 and Glu106 residues of TMV as discovered through matrix-assisted laser desorption/ionizationmass spectrometry and nuclear magnetic resonance spectroscopy c) Graphical schematic of the assembly of TMV spherical nanoparticles using a fluorous ponytail interaction (F-TMVCP). Cisplatin was loaded into the F-TMVCP using the same glutamic residues as in b (not shown) (reproduced with permission from ref [52] ).

Bacterial populations continue to evolve and escape current antibacterial treatments requiring improved antimicrobial approaches [56] . [58] . The VLPs were assessed for uptake both in vitro in RAW264.7 macrophages as well as in vivo in lung tissue from mice and were found to traffic into the RAW264.7 cells as well as to the lungs significantly greater than the negative control tolyl-labeled Qβ particles. As of yet, no efficacy studies have been reported.

Viruses are natural vehicles for delivery of genes. Because VLPs are devoid of their own genome, they can encapsulate nucleic acids easily and therefore have been broadly used for the delivery of genes as well as therapeutic nucleic acids. In fact, about 67% of gene therapy clinical trials world-wide are using viruses as vectors to transport genetic material [59] . However, traditional gene delivery strategies are based on mammalian viral vectors, and the application of plant viruses and phages is a newer iteration. While some reports indicate that cell targeting or cell penetrating peptides are not required to achieve gene delivery with plant VLPs as they are proteinaceous in nature imparting better cell penetration and endolysosomal escape than some synthetic nanoparticle systems [13] , a common strategy to enhance the delivery efficiency is the display of peptides that improve cell uptake and trafficking [60] . Plant viruses and phages did not evolve to circumvent mammalian cellular compartments and therefore to some degree become trapped within intracellular vesicles. 

Delivery of polypeptides and proteins requires different strategies as they can be more difficult to encapsulate. One strategy is chemical or genetic fusion of the A lot of recent work has been accomplished with encapsulating drug-activating enzymes within VLPs for therapeutic purposes [76] . Enzymes within the cytochrome P450 family are responsible for catalyzing chemotherapeutic prodrugs into their active forms, and encapsulating these enzymes within VLPs can reduce side effects, increase targeting to the tumor site, and boost retention within tumors via the enhanced permeability and retention effect [77, 78] . Initial results

show that a bacterial cytochrome, CYP BM3 , encapsulated within CCMV, could efficiently activate the pro-drugs tamoxifen and resveratrol [77] . An additional study with CYP BM3 -encapsulated bacteriophage P22 indicated that the enzyme-VLP therapy could enhance the activity of the CYP BM3 up to 10x that of endogenous levels when delivered with lipofectamine [79] . In addition to the delivery function, the VLP can offer protection against degradation by proteases.

P22-CYP BM3 and free CYP BM3 were incubated in trypsin for 1 hour; the CYP BM3 within the P22 retained up to 90.3% of its activity compared to 59.5% for the free CYP BM3 indicating protection from the protease.

In a different example, M13 bacteriophages were bioconjugated to horseradish peroxidase (on coat protein p9) and a peptide sequence called Ypep, which has targeting and penetration capability in PC-3 prostate cancer cells (on coat protein p3) [80] . [87] . The protein A coated PVX was then readily functionalized with IgG and was applied in biosensing of plant viruses [88] .

PVX can further be conjugated to fluorescent reporters to be used for in vivo imaging applications and theranostics [89] . PVX was engineered as a fluorescent probe through genetic manipulation to encode a small fluorescent protein called iLOV. Of note, iLOV display has also been applied to TMV [90] . To be used as a Our lab has also explored the application of TMV for cardiovascular therapy. In one example, we conjugated tissue plasminogen activator (TMV-tPA) to TMV;

tPA is a thrombolytic used in acute myocardial infarctions to break down thrombus obstructions [93] . Illumination of the right carotid artery of C57BL/6 mice with a 540 nm green laser produced thrombi. TMV-tPA was delivered via tail vein injection and maintained its efficacy and performed similarly to free tPA; more importantly, the TMV-tPA formulations displayed an improved safety profile indicated by a decreased average bleeding time of 429 seconds to 858 by the free tPA [94] . The decreased bleeding time is of noteworthy importance as tPA does not specifically congregate at the diseased area, and systemic administration can lead to life-threatening systemic hemorrhaging and/or angioedema [95] .

VLPs are powerful platforms for vaccines. VLPs mimic the conformation of native viruses harnessing their inherent immunogenicity but do not compromise on safety, as VLPs cannot infect nor replicate due to the absence of the viral genome [96] . properties are utilized to reprogram the tumor microenvironment (TME) to launch antitumor immunity. This section will discuss the recent progress within these fields.

The TME is highly immunosuppressive and favors tumor immune escape by suppressing the production, activation and function of antitumor T-cells [97] . As some VLPs are highly immunogenic, i.t. administration of VLPs into TMEs can trigger innate immune activation capable of instigating the transition to adaptive antitumor immunity [98] . In 2015, the first oncolytic viral immunotherapy named Imlygic, otherwise known as talimogene laherparepvec (TVEC), was approved by the Food and Drug

Administration [101] . TVEC is derived from type I herpes virus, but has been genetically modified to be unable to create the fever blisters seen in the infectious also induces systemic anti-tumor immunity [109] . The overall mechanism is outlined in Figure 5 . We further verified the immunotherapeutic efficacy of CPMV in separate studies in the treatment of murine dermal melanoma [110] , intracranial glioma [111] and ovarian cancer [109] . In addition to the mouse studies, we have begun testing the immunotherapy in canine patients, and our data indicate similar efficacy in these patients [112] . In the aforementioned CPMV studies, the immunotherapeutic vaccines are administered multiple times; however, multiple dosing regimens can lead to low patient compliance and increased hospital burden. Therefore, the VNPs of CPMV have also been applied as a slowrelease formulation by forming aggregates with polyamidoamine generation 4 dendrimers (CPMV-G4) [113] . As plant virus-based VLPs are stable, nontoxic, modifiable with drugs and antigens, and can be mass-manufactured [15] , this study has pioneered a new strategy of using plant virus-based VLPs for cancer immunotherapy.

We also tested the efficacy of other plant viruses as in situ vaccines. We found that while TMV showed some degree of efficacy, it could not match that reported for CPMV [110] . On the contrary, Lamarre et al. showed that i.t. administration of papaya mosaic virus (PapMV) could slow down B16-OVA melanoma progression and prolong survival [114] , through TLR7 activation induced by PapMV [98] .

Similar observations of therapeutic efficacy have been made using bacteriophages.

The M13 bacteriophage has been used for monotherapy against B16F10 melanoma cells triggering the immune response through TLR signaling [115] . Taken together, data indicate the potential of plant viruses and bacteriophages in tumor therapy, and one such formulation has already advanced to clinical testing.

The aforementioned Qβ formulation packaged with CpGs is currently in clinical trials for melanoma immunotherapy [70] .

J o u r n a l P r e -p r o o f 

Although i.t. administration of VLPs into the TME has shown potential to induce antitumor immune responses, cancer is inherently a heterogenous disease and even cells within the same tumor can showcase dissimilar responses to treatments [116] . CPMV has also been investigated in combination with radiation therapy (RT). RT leads to tumor debulking and tumor cell apoptosis, leaking a wide array of TAAs [121] . As mentioned above with the CPA studies, the CPMV promotes APC recruitment; the APCs phagocytose the extracellular TAAs leading to a sustained J o u r n a l P r e -p r o o f and improved response. Indeed, combining radiation therapy with CPMV in situ vaccination significantly improved tumor growth delay in ID8-Def29/Vegf-A-Luc tumors compared to either therapy alone [121] . The potent efficacy of CPMV + radiation therapy has also been translated to canine patients with melanoma [112] ;

therefore, this approach demonstrates high translational potential. 

As discussed in section 3.1, VLPs are immunogenic and are taken up by APCs;

recognition through PRRs leads to activation of the APCs and antigen processing.

VLPs comprised of multiple copies of their capsid proteins represent multivalent, repetitive scaffolds, which further benefit the multivalent antigen presentation.

These features make VLPs ideal platforms for epitope delivery and presentation to J o u r n a l P r e -p r o o f the immune system, and therefore VLP vaccines usually induce stronger immunity than antigens in their soluble forms. At the same time, the VLPs have adjuvant properties and many epitope display platforms do not require use of additional adjuvant to elicit potent immunity. We discuss the recent progress in using VLPs as carriers in vaccine design highlighting various application areas.

Several approaches of VLP-based vaccines are being considered: VLP subunit vaccines, viral vector vaccines, and VLP-based display platforms. Their mechanism of action is outlined in Figure 6 . Several viral vaccines are commercially available, these include human papillomavirus (HPV) and hepatitis B vaccines (HBV) and well as potential vaccines for influenza, Epstein-Barr virus, and malaria [126] [127] [128] [129] [130] .

J o u r n a l P r e -p r o o f Using the traditional methods of genetic insertion and chemical conjugation it can be difficult to obtain site-specific and highly efficient display of epitopes on VLPs.

To address this issue, an improved VLP display strategy via chemoenzymatic site- [131] . HBc VLPs were tagged through a sortase A-mediated transpeptidation reaction with an enterovirus 71 (EV71) SP70 epitope, which elicited effective antibodies against EV71 and protected Balb/c mice from lethal challenge. This chemoenzymatic site-specific approach showed great potential in VLP-based vaccine design for its simplicity, site specificity, high efficiency, and versatility.

Besides displaying antigens on the surface, VLPs can also encapsulate antigens within their interior and deliver antigens to immune cells for enhanced T-cell response (Figure 6 ). Douglas et al. developed a P22-based platform delivering both matrix and matrix 2 proteins derived from respiratory syncytial virus within the same particle [132] . Following intranasal administration in CB6F1/J mice, the vaccine stimulated CD8+ T-cell memory responses against both antigens, and the T-cells maintained tissue-resident memory phenotypes. Tissue-resident memory Tcells are critical in forming quick antiviral responses against re-exposure rapidly triggering both the innate and adaptive responses [133] .

Of the infectious disease vaccines being developed, human immunodeficiency virus (HIV) remains one of the major milestones to be achieved for vaccine development as there is still no vaccine that can induce broadly neutralizing J o u r n a l P r e -p r o o f antibodies (bNAbs) to prevent infection [134] . The only existing target for bNAbs is the envelope glycoprotein (Env), a trimer of heterodimers consisting of gp120 and gp41 subunits; however, subunit vaccines are oftentimes immunologically deficient. To improve the immunogenicity, Überla et al. generated Env VLPs containing helper T-cell epitopes derived from tetanus toxoid [135] . Preimmunization of Balb/c and C57BL/6J mice with the tetanus toxoid via intramuscular injection into the gastrocnemius muscle of the hind legs significantly increased Env-specific IgG titers mainly aided by the intrastructrual help of the helper T-cell epitopes.

Also, protein nanotechnology can enable next-generation vaccine development; for HIV, several protein cages have been utilized for subunit display. While the following examples are not VLPs, these protein cageslike VLPsare selfassembled from repeating protein units. In an early study, Env was genetically fused to a ferritin protein nanocage yielding a 24-subunit nanoparticle of 30 nm presenting 8 trimers [136] . Two following efficacy studies showed that Env trimers presented on ferritin elicited significantly higher binding of antibodies and therefore autologous neutralizing antibody responses than single trimers after immunization in rabbits [137, 138] . To help delineate how these vaccine nanoparticle candidates traffic in vivo, Irvine et al. generated a ferritin-Env J o u r n a l P r e -p r o o f presenting eight copies of a gp140 envelope trimer (MD39). They found that the ferritin-based Env immunogens were rapidly shuttled to a follicular DC network and then concentrated in germinal centers via a complement-, mannose-binding lectic-, and immunogen glycan-dependent manner [139] .

The aforementioned examples are from naturally occurring protein cages; recently, computational protein structure prediction research has fueled the design and Zealand White rabbits with I53-50-Env significantly increased the immunogenicity, inducing NAb responses, which were 40-fold higher compared to single Env immunizations. Furthermore, I53-50-Env VLPs exhibited superior efficacy to ferritin-Env VLPs in enhancing autologous NAb responses, most likely due to the high density of presented Env trimers on the I53-50.

As we are writing this article, a novel coronavirus (SARS-CoV-2) has spread throughout the world triggering globally mandated lockdowns and quarantines while institutions and pharmaceutical companies work towards the discovery of a J o u r n a l P r e -p r o o f novel and efficacious vaccine [141] . Of the vaccines under development, VLP, attenuated/inactivated, and viral vector vaccines are strong candidates. In fact, of the 90+ vaccines being developed, 5 vaccines are strictly VLP-based, 8 are inactivated/attenuated, and 25+ are viral vectors [142] . Medicago is one of the companies producing a VLP-based vaccine in plants using N. benthamiana for inplanta production of empty SARS-CoV-2 viral particles [143] . The University of Wisconsin-Madison/Flugen/Bharat Biotech's live-attenuated influenza vaccine [144] or Sinovac's formalin-inactivated SARS-CoV-2 viral vaccine [145] , which has recently moved into Phase1/2 clinical trials [146] , also remain strong candidates.

VLPs have also been widely explored as a platform to deliver TAAs for use as cancer vaccines. Genetic fusion, chemical conjugation techniques, as well as enzyme-mediated ligation strategies have been utilized to attach TAAs onto VLPs.

For cancer vaccines, a particular challenge is to break tolerance for self-antigens. Chemical conjugation is the most commonly utilized method for attachment of TAAs to VLPs, and our group has been utilizing diverse plant virus-based nanotechnology platforms for the delivery of TAAs. The comparison of icosahedral CPMV and filamentous VNPs for human epidermal growth factor receptor 2 (HER2) epitope delivery revealed that the icosahedral CPMV was more efficient in terms of lymphatic drainage, and uptake by and activation of APCs, leading to enhanced anti-HER2 immunity compared to filamentous PVX [148, 149] .

The CPMV-HER2 vaccine candidate delayed the progression of DDHER2 primary tumors and metastasis in Balb/c mice thereby prolonging survival [150] . The displayed epitope plays an integral part in the immunotherapeutic response, but the nanocarrier itself can also affect the effectiveness of the therapy. In a recent study, we compared three icosahedral plant VNPs, CPMV, CCMV and sesbania mosaic virus (SeMV), displaying a HER2 epitope as a cancer vaccine [151] . Although the three viruses are similar in shape and structure, there were significant differences between the immune profiles elicited by the viruses. CPMV-HER2 induced a Th1 predominant immune response while CCMV-and SeMV-HER2 induced a Th2 Although chemical conjugation remains the most widely used methodology for attachment of antigens to VLP surfaces, specific ligation strategies such as the previously mentioned Spy-tag/Spy-catcher conjugation can confer additional spatial regulation and provide increased stoichiometric control of antigen presentation. Acinetobacter bacteriophage AP205 derived VLPs were engineered in this fashion to display multivalent HER2 epitopes on the surface [152] .

Immunization studies indicated that the vaccine overcame B-cell tolerance and induced strong polyclonal anti-HER2 response.

Aside from breast cancer, cervical cancer is responsible for the most cancer-related deaths in women worldwide [153] . Cervical cancer is mainly caused by HPV, and recent studies have utilized plant VLPs and bacteriophage as potential vaccines. high antibody specificity to the Tn. In a follow-up study, TMV was used to display Tn antigens [162] . Unlike with CPMV, the location of the Tn conjugation was an important factor in IgG production; Tn linked to the N-terminus of TMV was immunosilent, but conjugation to Tyr 139 elicited strong immune responses.

Alternatively, tumor associated neoantigens can produce more specific antitumor immune responses and have the potential to be used in personalized vaccines [163] .

A multi-target vaccine strategy using Qβ VLPs containing TLR ligands was evaluated against B16F10 murine melanoma. The study compared vaccines containing germline epitopes identified through immunopeptidomics, mutated epitopes predicted by whole exome sequencing, and a third combining both germline and mutated epitopes [164] . Pre-clinical evaluation indicated that the Qβbased vaccines presenting both the germline and mutated epitopes achieved the greatest therapeutic efficacy.

Lastly, nucleic acid vaccines have garnered strong interest as next-generation cancer vaccines, and VLPs have been explored as potential carriers. RNA is instable in vivo due to the presence of RNases; thus, nanocarrierssuch as VLPscan help protect and extend the biodistribution and half-lives of these therapies. [165] . Following administration, GM-CSF production was measured as early as 12 hours after APC phagocytosis in vitro. Fluorescence activated cell sorting of DC markers confirmed DC activation and maturation leading to strong humoral and cellular immune responses. The vaccine was administered i.v., which protected C57BL/6 mice against TRAMP-C2 prostate cancer prophylactically, and as a therapeutic vaccine, retarded tumor growth compared to soluble GM-CSF and VLP-only controls.

VLP vaccines can also be used to treat a range of other disorders, such as neurological, cardiovascular and autoimmune diseases. Currently, there are no approved vaccines for tauopathies such as frontotemporal dementia (FTD) and

Alzheimer's disease (AD). These diseases cause unrecoverable cognitive decline and lower quality of life extremely for patients. Data suggests that tauopathies occur due to aggregation of amyloid beta (A) peptides as well as the accumulation of microtubule-associated protein tau (pTau) within neurons forming neurofibrillary tangles [166] . Therefore, tauopathy vaccines should target either A or pTau and produce long-lived antibody responses against these proteins. AD vaccines against A fail in the clinic due to strong T-cell responses from the J o u r n a l P r e -p r o o f vaccine and the simultaneously administered adjuvant leading to meningoencephalitis and self-tolerance to the anti-A antibodies [167] . To counteract these deficiencies, Morgan et al. developed Qβ-derived VLPs conjugated on their N-and C-termini to A peptide sequences [167] . The Nterminal vaccine mainly elicited an IgM response while the C-terminal vaccine elicited an IgG responsehighlighting an inherent need in increasing understanding in design rules that govern the engineering of effective vaccines.

Regardless, both vaccines elicited strong antibody titers without the need for an adjuvant in an amyloid precursor protein mouse model and reduced amyloid plaques in the cortex significantly. In a later study, Bhaskar et al. engineered Qβderived VLPs to deliver multivalent tau peptide with phosphorylation at threonine 181 (pT181) [168] . Bi-weekly i.m. vaccination in rTg4510 mice models indicated that the vaccine candidate induced a robust and long-lived anti-pT181 antibody response leading to reduction of phosphorylated pathological tau in the hippocampus and cortex, prevention of atrophy in the hippocampus and corpus callosum, and rescue of cognitive dysfunction. It is also worth mentioning that mammalian VLPs such as HBc [169] , murine leukemia virus [170] , and HPV [171] have been explored with relative success as tauopathy vaccines.

VLPs have also been investigated as vaccines for inflammatory and autoimmune diseases. IL-17 mediates the release of pro-inflammatory cytokines in many cells, and IL-17 producing T helper cells (Th17) are implicated in several autoimmune disorders, such as rheumatoid arthritis and multiple sclerosis [172] . Monoclonal IL-17 antibody blockade has shown previous treatment efficacy, and thus one alternative strategy is the development of vaccines that can produce anti-IL-17 antibodies [173] . For this purpose, Qβ-based VLPs carrying IL-17 were developed;

the VLPs overcame self-tolerance against IL-17 and generated high titers of anti-IL-17 antibodies to slow the progression of autoimmune arthritis and encephalomyelitis [174] . Another inflammatory disease being considered for vaccine development is allergic asthma; the disease is induced by chronic inflammation in the lungs, which is mainly mediated by the recruitment and activity of eosinophils [175] . IL-5 and eotaxin are chemokines vital for the recruitment and maturation of eosinophils from the bone marrow [176] . Q particles were chemically crosslinked to either IL-5 or eotaxin, and immunization with these particles overcame self-tolerance and stimulated high antibody response against the two particles. Co-immunization with Q-IL-5 and Q-eoxtaxin lowered eosinophil-induced inflammation in Balb/c mice. Allergic airway inflammation in the mice was induced through the administration of ovalbumin and alum [176] .

Another target application of engineered VLP vaccines lies in the treatment of cardiovascular diseases (CD). Mutations that increase the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that controls cholesterol homeostasis by enhancing the degradation of the low-density lipoprotein receptor (LDL-R), can lead to hypercholesterolemia, atherosclerosis, and early-onset CD [177] . Qβ-based VLPs were engineered to display PCSK9derived peptides, and subsequent vaccination in mice and macaques elicited high titers of PCSK9-specific antibodies thereby inhibiting the enzyme and lowering the levels of cholesterol, phospholipids and triglycerides [177] . The VLP also worked synergistically with simvastatin in lowering LDL levels. The pathophysiology of atherosclerosis can also be attributed to overproduction of the proinflammatory cytokine IL-1 [178] . Vaccines targeting IL-1 should therefore produce NAbs against the cytokine, effectively disrupting its function. Bachmann et al. developed a Qβ-based vaccine with conjugated full-length IL-1, and vaccination in ApoEdeficient mice fed a Western diet led to significant anti-IL-1 titers in 21 days [179] . Immunization further led to a 50 and 37% decrease of plaque progression in the aorta and aortic root respectively, 22% decrease in macrophage infiltration, and decreased levels of the pro-inflammatory molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1).

Lastly, VLPs have been investigated as vaccines against pain and as contraceptives.

Nerve growth factor (NGF) is a key cause underlying the chronic pain associated with osteoarthritis [180] . Cucumber mosaic virus (CuMV) was used as a scaffold for conjugation of NGF to its exterior surface. The CuMV-NGF VLPs induced high titers of anti-NGF antibodies in mice that had undergone partial meniscectomy-induced osteoarthritis, leading to the reversal of pain behavior [181] .

As a contraceptive, Johnson grass mosaic VLPs displaying the spermatozoa peptide YLP12 and zona pellucida epitope ZP3 elicited antibodies that block the ability of the sperm to interact with the zona pellucida of the egg [182] . s.c.

immunization in FVB/cJ mice with the contraceptive VLPs decreased litters born compared to controls. The far-ranging applicability of VLPs in neurological, cardiovascular, and autoimmune diseases as well as in pain and contraception highlight the flexibility of VLPs as vaccination agents and evidences the justified advancement in the field for future vaccine strategies.

Molecular imaging is a growing biomedical research discipline that enables the visualization, characterization and quantification of biological processes in vivo. have been widely used to depict cellular and molecular pathways and mechanisms of disease present in the context of living subjects (Figure 7) . Ideal molecular imaging techniques should quickly provide optimal signal-to-noise ratios at the target site with minimal toxicity. Compared to synthetic nanoparticles, VLPs can be more advantageous as imaging delivery agents because of their short circulation and retention time decreasing possible side effects [183] . Moreover, VLPs can be tailored to carry a wide range of fluorescent labels and/or contrast agents while being modified with aptamers, peptides or antibodies for improved targeting to particular cells and tissues. In this section, we will summarize the VLP-based platforms for delivery of imaging agents. 

Fluorescent VLPs are used in optical imaging for specific labeling of cells and tissues in addition to analyzing particular biodistributions, pharmacokinetics and biological interactions [148, 184] . The plant virus, CPMV, has been extensively used for optical imaging applications. A unique feature of CPMV is that it naturally interacts with the mammalian protein surface vimentin and this enables imaging of the endothelium [185] , and in particular the tumor endothelium [186] .

In an early study, the tumor neovasculature was imaged with fluorescent CPMV VNPs using intravital two-photon laser scanning microscopy (TPLSM) [185] .

Future studies explored engineered VNPs with added receptor specificity: for example, CPMV was modified with a peptide ligand, E7p72, which has high affinity for epidermal growth factor-like domain 7 (EGFL7) [187] . The E7p72-CPMV particles showed high specificity to EGFL7 and demonstrated binding to tumor neovasculature within 90 minutes as seen through real-time intravital imaging. While native CPMV also showed interaction with the endotheliumthis was much less profound due to non-specific macrophage clearance. In the CPMV studies, fluorescence was introduced through chemical coupling of near-infrared (NIR) fluorophores. However, genetic engineering approaches have also been exploited to develop fluorescent VLPs. In section 2.5, we already discussed PVX and TMV engineered to express the small fluorescent protein called iLOV [90] J o u r n a l P r e -p r o o f amongst other examples. Further, our lab has constructed PVX VNPs produced in N. benthamiana plants that have been genetically engineered to display GFP or mCherry at their N-termini [188] . The PVX displayed 1 fluorescently-modified coat protein for every 3 wild-type coat proteins. The fluorescent PVX was used to track the viral infection process in plants, to optically image HT-29 cells in vitro, and for particle tracking in vivo using a HT-29 murine model in C57BL/6 mice.

Besides cancer imaging, VLPs have been engineered and studies in the context of CD imaging. One application has focused on the design of VLPs loaded with contrast agents to stratify atherosclerotic plaques, thus improving the accuracy of prognoses and reducing the frequency of heart attacks and strokes. MS2 VLPs have been utilized to carry NIR fluorescent dyes (to enable imaging) and anti-VCAM-1 antibodies (to enable targeting of the sites of inflammation) [189] . The study demonstrated that the engineering VLPs were effective to target and detect atherosclerotic plaques of different sizes in the descending aorta and aortic arch in ApoE knockout mice while untargeted controls showed limited signs of fluorescence in the plaques. Our lab has focused on the study and application of TMV for CD imaging applications targeting both atherosclerosis (early stage disease) and thrombosis (late stage disease). For example, TMV was conjugated to both an NIR dye and a peptide targeting S100A9 (aka myeloid-related protein 14) J o u r n a l P r e -p r o o f [190] . S100A9 is a molecular marker found in atherosclerotic lesions that are prone to rupture and was identified as one of the most promising potential markers for acute myocardial infarctions [191, 192] . The targeted TMV particles were injected i.v. and detected the presence of macrophage-rich atherosclerotic lesions in ApoE -/mice while untargeted TMV controls showed insignificant detection capabilities enabling the platform's possible use in the future as a marker for at-risk lesions.

The two studies above solely targeted single proteins (e.g. VCAM-1 and S100A9), but targeting different molecules may provide additional information such as the extent of progression of CD. Toward this goal, a trifunctional VLP platform derived from SV40 carrying NIR quantum dots, a targeting peptide, and an anticoagulant drug (Hirulog) was developed for in vivo targeting, imaging and treatment of atherosclerotic plaques [193] . Depending on the targeting peptide used (against VCAM-1, macrophages, or fibrin), the VLP could detect atherosclerosis at different stages of CD (early, developmental, and late respectively). Furthermore, the delivery of the Hirulog to aortic atherosclerotic plaques led to increased antithrombotic activity compared to nontargeted VLPs.

Lastly, fluorescent reporters can oftentimes be easily degraded within the body leading to diminished imaging capabilities. To improve imaging times, VLPs can be incorporated into long-lasting formulations using slow-release polymers, J o u r n a l P r e -p r o o f scaffolds, and metal-organic frameworks (MOFs). A recent paper by Gassensmith et al. details a TMV-MOF hybrid nanoparticle that increases TMV VNP retention when delivered s.c. into Balb/c mice [194] . Cy5-encpsulated TMV is coated with zeolitic imidazolate framework-8 (Cy5-TMV@ZIF), and when compared to native Cy5-TMV, the Cy5-TMV@ZIF retained its fluorescence for ~2.5x the time of Cy5-TMV (288 hours vs. 120 hours). Histology studies showed no signs of toxicity due to the MOF. The TMV nanoparticles were also stressed in harsh conditions through heating at 100C for 20 min or soaking in methanol or 6M guanidinium chloride overnight. While WT TMV showcased significant deterioration, the TMV@ZIF remained stable.

Optical imaging techniques are considered safe and do not expose patients to harmful radiation, but they are usually limited to 1-2 mm of tissue penetration and are restricted translationally [195] . On the contrary, MRI, PET, and CT are used extensively in the clinic and can provide extremely accurate images deep within tissues. VLPs can be loaded with MRI, PET or CT contrast agents and these approaches are discussed in the following sections. VLPs can be loaded with hundreds-to-thousands of copies of contrast agents; this increases the local concentration and therefore the signal-to-noise ratio particularly when these J o u r n a l P r e -p r o o f nanoparticles are engineered to target specific tissues and cells. When it comes to MRI application, the protein-based contrast agents also confer advantages in reducing residence time of the contrast agents within tissues, therefore lowering the risk of toxicity. For example, contrast agents such as gadolinium (Gd) can be quite toxic; but VLPs are generally cleared rapidly from circulation and tissues therefore reducing systemic toxicity [196] . In early work, we demonstrated that TMV loaded with Gd-dodecane tetraacetic acid (Gd-DOTA) and modified to target VCAM-1 enabled sensitive detection and delineation of atherosclerotic plaques in ApoE -/mice using submicromolar doses of the contrast agent [197] . The high aspect ratio of TMV promoted ligand-receptor interactions to enhance margination to the diseased vessel wall, and the slower tumbling and enhanced relaxivity of the Gd-DOTA coupled to the macromolecular carrier increased the signal-to-noise ratio. The TMV platform increased the imaging sensitivity considerably allowing for a 400x reduction in Gd dose compared to standard clinical concentrations.

Given their high degree of surface functionality offering distinct amino acid residues on both their internal and external surfaces, VLPs make excellent platforms for development of multi-modal imaging probes. In one example from our lab, we demonstrated the loading of a dysprosium (Dy 3+ ) complex in the internal cavity of TMV to increase T2 relaxivity for MRI; and the simultaneous delivery of NIR fluorescent dyes enabled dual optical-MR imaging. Target J o u r n a l P r e -p r o o f specificity was achieved by labeling the external surface of TMV with an Asp-Gly-Glu-Ala peptide to target integrin α 2 β 1 on prostate cancer cells. Feasibility of this dual-modal imaging approach was demonstrated in athymic nude mice with PC-3 prostate cancer xenografts [198] . While most VLPs exhibit short circulation times and rapid tissue clearance (~ 3 days); we discovered that the VLPs from PhMV allow imaging of tumors over extended periods of time (~ 10 days). In this study, PhMV was modified identically to the aforementioned TMV probe: PhMV was loaded with a NIR dye for optical imaging, Gd-DOTA for MRI, and the Asp-Gly-Glu-Ala targeting peptide; and longitudinal imaging of prostate tumors in vivo was demonstrated [184] . The long residence time within tumors may be utilized to follow disease progression and therapeutic responses.

Outside of plant virus nanotechnology, Kang et al. has utilized P22 bacteriophages for intravascular MRI imaging [199] . Here, paramagnetic Gd ions complexed to chelating agent diethylenetriamine pentaacetic acid (DTPA) were conjugated to the interior and exterior of the P22 capsids (P22-Gd-DTPA) using maleimide chemistries. When administered into Balb/c nude mice, the P22-Gd-DTPA contrast agent enabled imaging of the carotid, mammary arteries, jugular vein, and superficial vessels at a resolution of 250 m (it should be noted that non-targeted P22 was utilized). Using the P22 platform, work has also been done to increase the J o u r n a l P r e -p r o o f contrast agent loading capacity through use of polymerization chemistry [200] .

Here instead of conjugating the contrast agent to the surface of the capsid; a polymer network is formed within the capsid and the contrast agent is incorporated within, therefore enhancing loading capacity. Specifically, a 2-aminoethyl methacrylate (AEMA) polymer was synthesized within P22 capsids using ATRP, enabling loading with up to 3,646 paramagnetic manganese (III) protoporphyrin IX (MnPP) sites [201] . The resulting ionic relaxivities were comparable to free MnPP, but initial studies suggest the relaxivity could be improved by reducing the intramolecular interactions between MnPP molecules.

Contrast agents in the clinic have been mainly restricted to paramagnetic metal complexes such as Gd and Mn; however, newer generation contrast agents can improve resolution and additionally provide functional information. These newer contrast agents utilize mechanisms such as chemical exchange saturation transfer (CEST) to improve upon current standards [202] . As a proof-of-principle with VLPs, filamentous fd bacteriophages expressing anti-EGFR were transaminated to introduce ketone groups followed by oxime ligation of a cage-like xenon-binding molecule (CryA) [203] . 129 In another study, TMV loaded with a metal-free paramagnetic nitroxide organic radical contrast agent (ORCA) for MRI and electron paramagnetic resonance probes for superoxide detection showed enhanced in vitro r1 and r2 relaxivities [204] . The probes functioned as both T1 and T2 contrast agents making this platform suitable for pre-clinical and clinical MRI scanners. Furthermore, by conjugating fluorescent dyes on the internal surface and ORCA on the outer surface, the TMV probes could be used as a concentration marker regardless of the ORCA oxidation state. In a more recent study by the same group, TMV was conjugated to a derivative of the aminoxyl radical TEMPO (coined Compound 6) by a copper-catalyzed azide-alkyne cycloaddition reaction (TMV-6) [205] . Subsequent binding to cucurbit [8] uril (CB [8] ) forms an ORCA inclusion complex called a semirotaxane that helps shield the aminoxyl radicals from reduction into hydroxylaminesa process which makes aminoxyl-based ORCAs MRI-silent.

Measurements for r 1 values of TMV-6 rivaled that of Gd-DOTA while the semirotaxane also showed an order of magnitude increase in r 1 compared to CB [8] and 6 alone. Most importantly, in the presence of sodium ascorbate, TMV-6 was reduced at a half-life of 7.2 minutes while the semirotaxane showcased half-lives of 577.6 minutes, an increase in two orders of magnitude.

J o u r n a l P r e -p r o o f

While MRI works on the structural level, PET can image at the metabolic level for refined imaging of pathophysiological changes within the human body [206] . PET is mainly used in the clinic for oncological imaging as cancerous cells undergo metabolic changes that PET scans can detect much earlier than CT or MRI. PET is oftentimes combined with CT imaging to provide both structural and metabolic information of tissues within the patient. One example by Francis et al. was the engineering of DOTA-MS2 bacteriophages to deliver 64 Cu for microPET/CT imaging [207] . Compared to free 64 Cu, which was cleared rapidly, the 64 Cu-labeled DOTA-MS2 phages lasted in circulation for over 24 hours at > 20% injected dose (ID)/gram although specific tumor homing was not evidenced. To improve the specificity of the PET imaging modalities and improve tumor homing, one can attach targeting ligands. This was demonstrated with RGD peptide-conjugated T7 [208] and anti-EGFR conjugated MS2 bacteriophages [82] . The T7 phages were genetically engineered to express solvent-exposed RGD sequences followed by the chemical addition of 64 Cu-DOTA. These phages were able to specifically extravasate into U87MG tumor xenografts displaying up to 10% ID/gram. When cold RGD peptide was added before T7 administration (for receptor blocking), tumor uptake was significantly reduced verifying the specificity of the PET imaging. Also, the EGFR-conjugated MS2 bacteriophages showed up to 10-15% J o u r n a l P r e -p r o o f ID/gram 24 hours after injection in plasma and up to 2-5% ID/gram within HCC1954 orthotopic breast cancer tumors [82] .

PET is not only utilized in solid tumor imaging, but also can be used to track micrometastases in sentinel lymph nodes [209] . NV1023 is an oncolytic herpes Although CT is oftentimes used concurrently with PET, it can also be employed as a stand-alone imaging modality. Recently, gold-coated VNPs were developed using CPMV. These materials were engineered for CT imaging using scattered Xray for 3D visual reconstruction and tissue segmentation [210] . The gold-coated CPMV improved the sensitivity of the technique by reducing the scan time to less than 2 min and achieved a resolution of nearly 150 Hounsfield units.

J o u r n a l P r e -p r o o f

In newer generation imaging applications, VLPs can be simultaneously loaded with imaging reagents and drugs; and therefore, be used for both diagnosis and therapy -a concept known as theranostics. One of the most promising strategies is PTT, which employs NIR laser photoabsorbers to generate heat for thermal ablation of cancer cells upon laser irradiation [211] . Compared with conventional therapeutic modalities, PTT exhibits unique advantages in cancer therapy such as high specificity, minimal invasiveness, and precise spatial-temporal selectivity [212, 213] . Qβ-based VLPs were engineered for PTT through the attachment of sub 7-nm sized gold nanoparticles on their surface; the inner cavity was loaded with upwards of 500 DOX molecules [214] . Upon 6-nanosecond laser irradiation, DOX was rapidly released from the capsid without heating the bulk solution, allowing for highly targeted cell killing of RAW264.7 macrophage and A549 lung cancer cells in vitro. Live-cell imaging indicated that only cells inside the irradiated area showed morphological alterations and detachment signifying highly localized treatment capabilities. Another study with Qβ conjugated to metalloporphyrin derivatives and a CD22 receptor targeting glycan was utilized in photodynamic therapy (PDT) [215] . In vitro studies with CD22 positive Chinese hamster ovary cells indicated dose-responsive phototoxicity and greater cell killing in targeted vs.

J o u r n a l P r e -p r o o f non-targeted viral particles. PDT has additionally been utilized with P22 VLPs encapsulating CYP (which converts the prodrug tamoxifen) and decorated with the photosensitizer protoporphyrin IX against estrogen receptor positive breast cancer cells [216] . To confer MRI and photoacoustic imaging capabilities to the PTT treatment for use in theranostics, the nanorods of TMV were used to load Gd and coated with polydopamine (PDA); the PDA coating enhances the MRI properties and confers PDA contrast while enabling PTT [217] . MRI imaging of Gd-TMV nanoparticles without PDA produced r 1 values of up to ~13 mM -1 s -1 , better than the r 1 values of commercial MRI contrast agents (3-5 mM -1 s -1 ). PDA coating onto the TMV (Gd-TMV-PDA) led to far greater r 1 values, reaching up to ~ 80 mM -1 s -1 .

This increase in relaxivity may be explained by the fact that the PDA coating traps bulk water in the structure of TMV therefore further enhancing the magnetic properties and interaction of the Gd centers with water. Furthermore, the Gd-TMV-PDA particles showed strong NIR absorption in vitro with high photothermal conversion efficiency (28.9%). In comparison, other commonly used photothermal agents report photothermal conversion efficiencies of 24.4% (Fe@Fe 3 O 4 ) [218] , 13% (gold nanocages) [219] , 21% (gold nanorods) [220] , and 22% (Cu 2-x Se) [221] . The nanoparticles showed strong efficacy in vitro in treating PC-3 prostate and 4T1 breast cancer cells while the controls of cells + irradiation and cells + Gd-TMV-PDA without irradiation showed minimal cytotoxicity.

Mammalian viruses have also been utilized in PTT. HBc has been used to encapsulate copper sulfide for PTT only [222] , conjugated to a RGD peptide for tumor targeting and loaded with indocyanine green for image-guided PTT [223] , and loaded with iron oxide and methotrexate for MRI and PTT [224] . Lastly, ferritin based VLPs have been used to load copper sulfate [225] and gold nanoparticles [226] for cancer theranostics.

In some more advanced theranostic modalities, the imaging reagents and drugs can be activated simultaneously. The bacteriophage M13 was conjugated with DOX, a fluorophore, and a targeting ligand to secreted protein, acidic, and rich in cysteine (SPARC), which is overexpressed in melanoma, breast, brain, prostate, colon, and lung cancers [227] . The multifunctional M13 nanoparticles were tested using two prostate cancer lines: one with high SPARC expression (C4-2B) and one with low SPARC expression (DU145). The IC 50 curves indicated strong cytotoxicity towards the C4-2B cell line, but not the DU145 cells, demonstrating specificity towards SPARC. Fluorescence images showed greater cell adherence of the VLPs towards the C4-2B cell line.

J o u r n a l P r e -p r o o f

The field of viral drug delivery has taken tremendous steps forward in recent years; this includes the application of plant viruses for treatment of canine cancer patients [112] , clinical trials using CpG-laden bacteriophages [70] , as well as adenoviral COVID-19 vaccine candidates that are currently in phase 2 clinical testing [228, 229] . These examples highlight that the field has grown out of its infancy.

However, there remain challenges in further advancing the viral drug delivery field.

The most obvious hurdle is whether pre-existing or de novo acquired adaptive immunity against the viral carrier would lead to adverse effects or reduce efficacy upon repeat administration [230] . This may be a challenge for traditional drug delivery approaches targeting diseased cells and tissueshowever, surface passivation or immune editing methods have shown promise [231] [232] [233] . When it comes to immunotherapy applications, the immunogenicity of the carrier is less likely to be a barrier. Rather, opsonization of the carrier primes the VNP for immune cell uptake, therefore boosting efficacy rather than interfering with it. This was validated for both in situ vaccine applications and mRNA vaccines delivered by the plant viruses CPMV and CCMV respectively [67, 234] .

As the field matures and more products enter clinical testing and development, efforts must focus on improving process development and large-scale J o u r n a l P r e -p r o o f manufacturing techniques. Lab protocols often involve centrifugation/ultracentrifugation for purification of the viruses or VLPs, but these processes are generally not scalable and require adaptation to industry standards (e.g. chromatography). Also, attention should be paid toward formulation quality control and assurancewhen VLPs are produced by (heterologous) expression they are devoid of their genome; however, they can oftentimes package host RNA causing unwanted side effects [235] . Mammalian viruses may also cause horizontal genetic transfer events evolving both humans and viruses in unimaginable ways [236] . These possibilities must be carefully examined and studied as well as regulated.

Lastly, from a regulatory perspective, while mammalian viral vectors and VLP subunit vaccines have long been tested for clinical application, with several formulations already approved and licensed, bacteriophage-and plant virus-based therapeutic approaches are just at the horizon to enter clinical development stages. This is an exciting time for the field, and we hope to see emerging viral nanotechnologies soon make the bench-to-bed transition.

J o u r n a l P r e -p r o o f

As highlighted throughout this article, plant viruses and bacteriophages are recognized nanotechnologies, which have been engineered for diverse applications in nanomachine (Figure 8) . VLPs have evolved to carry and deliver cargo making them natural experts at drug delivery. Most VLPs can withstand harsh environments, which allows their functionalization and engineering in the test tube; yet, VLPs are biocompatible and biodegradable. The engineering design space is unparalleled enabling genetic programming, self-assembly and chemical biology approaches to be utilized yielding functionalized hybrid VLP nanomaterials.

Furthermore, the structures of many viruses are understood at atomic resolution allowing modifications with precise spatial selectivity. The shape and size of VLPs facilitate their vascular transport, cellular uptake and interactions. Large doses of VLPs are usually well tolerated, but proteolytic degradation ensures their rapid and complete clearance for reduced side effects. Moreover, VLPs can be easily engineered to generate new structures that may interact predictably with biological systems; VLPs can display functional groups that include targeting ligands, imaging dyes, epitopes, or they can carry a payload of drugs or dyes to specific cells and tissues. The field of VLPs for drug delivery applications has come a long way since its introduction. As the field continues to grow and flourish, the number 

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Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle

novel coronavirus (2019-nCoV) outbreak: A new challenge

The race for coronavirus vaccines: a graphical guide

Medicago Develops Plant-Based Coronavirus Vaccine Candidate

Sinovac Biotech to move ahead with COVID-19 vaccine trial in China

Safety and Immunogenicity Study of Inactivated Vaccine for Prophylaxis of SARS CoV-2 Infection (COVID-19) -Full Text View -ClinicalTrials.gov

Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns

Multiple Administrations of Viral Nanoparticles Alter in Vivo Behavior-Insights from Intravital Microscopy

Plant viral nanoparticles-based HER2 vaccine: Immune response influenced by differential transport, localization and cellular interactions of particulate carriers

A Viral Nanoparticle Cancer Vaccine Delays Tumor Progression and Prolongs Survival in a HER2+ Tumor Mouse Model

Heterologous Prime-Boost Enhances the Antitumor Immune Response Elicited by Plant-Virus-Based Cancer Vaccine

Virus-like particle display of HER2 induces potent anti-cancer responses

Update knowledge on cervical cancer incidence and prevalence in Asia, Asian Pac

VLPs Displaying a Single L2 Epitope Induce Broadly Cross-Neutralizing Antibodies against Human Papillomavirus

Plant virus particles carrying tumour antigen activate TLR7 and Induce high levels of protective antibody

Boosting Immunity to Small Tumor-Associated Carbohydrates with Bacteriophage Qβ Capsids

Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies

Protective Epitope Discovery and Design of MUC1-based Vaccine for Effective Tumor Protections in Immunotolerant Mice

Synthesis and Immunological Evaluation of Disaccharide Bearing MUC-1 Glycopeptide Conjugates with Virus-like Particles

Antitumor Humoral and T Cell Responses by Mucin-1 Conjugates of Bacteriophage Qβ in Wild-type Mice

Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines

Tobacco mosaic virus as a new carrier for tumor associated carbohydrate antigens

Personalized vaccines for cancer immunotherapy

Targeting Mutated Plus Germline Epitopes Confers Pre-clinical Efficacy of an Instantly Formulated Cancer Nano-Vaccine

Messenger RNA vaccine based on recombinant MS2 virus-like particles against prostate cancer

National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease

Virus-like Peptide Vaccines Against Aß N-terminal or C-Terminal Domains Reduce Amyloid Deposition in APP Transgenic Mice without Addition of Adjuvant

Qß Virus-like particle-based vaccine induces robust immunity and protects against tauopathy, NPJ Vaccines

Hepatitis B core VLP-based mis-disordered tau vaccine elicits strong immune response and alleviates cognitive deficits and neuropathology progression in Tau.P301S mouse model of Alzheimer's disease and frontotemporal dementia

Vaccination with Abeta-displaying virus-like particles reduces soluble and insoluble cerebral Abeta and lowers plaque burden in APP transgenic mice

Overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of B cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice

Regulation of IL-17 in autoimmune diseases by transcriptional factors and microRNAs

Anti-IL-17 monoclonal antibodies for the treatment of ankylosing spondylitis

Vaccination against IL-17 suppresses autoimmune arthritis and encephalomyelitis

The Prospects of an Active Vaccine Against Asthma Targeting IL-5, Front Microbiol

Combined vaccination against IL-5 and eotaxin blocks eosinophilia in mice

A Cholesterol-Lowering VLP Vaccine that Targets PCSK9, Vaccine

IL-1 and atherosclerosis: a murine twist to an evolving human story

A VLP-based vaccine against interleukin-1α protects mice from atherosclerosis

NERVE GROWTH FACTOR (NGF) BLOCKADE FOR THE MANAGEMENT OF OSTEOARTHRITIS PAIN: WHAT CAN WE LEARN FROM CLINICAL TRIALS AND PRECLINICAL MODELS?

Active immunisation targeting nerve growth factor attenuates chronic pain behaviour in murine osteoarthritis

Immunogenicity of zona pellucida glycoprotein-3 and spermatozoa YLP(12) peptides presented on Johnson grass mosaic virus-like particles

Viral nanoparticles as platforms for next-generation therapeutics and imaging devices

Physalis Mottle Virus-like Nanoparticles for Targeted Cancer Imaging

Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles

Cowpea mosaic virus nanoparticles for cancer imaging and therapy

Viral nanoparticles decorated with novel EGFL7 ligands enable intravital imaging of tumor neovasculature

Planta Production of Fluorescent Filamentous Plant Virus-Based Nanoparticles

Vascular Cell Adhesion Molecule-Targeted MS2 Viral Capsids for the Detection of Early-Stage Atherosclerotic Plaques

S100A9-targeted tobacco mosaic virus nanoparticles exhibit high specificity toward atherosclerotic lesions in ApoE −/− mice

Platelet Expression Profiling and Clinical Validation of Myeloid-Related Protein-14 as a Novel Determinant of Cardiovascular Events

High neutrophil numbers in human carotid atherosclerotic plaques are associated with characteristics of rupture-prone lesions

Vivo Targeting and Imaging of Atherosclerosis Using Multifunctional Virus-Like Particles of Simian Virus 40

Enhanced Stability and Controlled Delivery of MOF-Encapsulated Vaccines and Their Immunogenic Response In Vivo

Van den Abbeele, Chapter 6 -Imaging Tools in Human Research

Gadolinium-based contrast agent toxicity: a review of known and proposed mechanisms

Dual-modal magnetic resonance and fluorescence imaging of atherosclerotic plaques in vivo using VCAM-1 targeted tobacco mosaic virus

Dysprosium-Modified Tobacco Mosaic Virus Nanoparticles for Ultra-High-Field Magnetic Resonance and Near-Infrared Fluorescence Imaging of Prostate Cancer

Implementation of P22 Viral Capsids As Intravascular Magnetic Resonance T1 Contrast Conjugates via Site-Selective Attachment of Gd(III)-Chelating Agents

Manganese(III) porphyrins complexed with P22 virus-like particles as T1-enhanced contrast agents for magnetic resonance imaging

Using the Interior Cavity of the P22 Capsid for Site Specific Initiation of Atom Transfer Radical Polymerization with Tremendously Increased Cargo Loading

Paramagnetic lanthanide complexes as PARACEST agents for medical imaging

Molecular imaging of cancer cells using a bacteriophage-based 129Xe NMR biosensor

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor

Supramolecular and biomacromolecular enhancement of metal-free magnetic resonance imaging contrast agents

Clinical Applications of PET and PET-CT

PET Imaging and biodistribution of chemically modified bacteriophage MS2

Trackable and Targeted Phage as Positron Emission Tomography (PET) Agent for Cancer Imaging

Imaging of lymph node micrometastases using an oncolytic herpes virus and [F]FEAU PET

Gold-coated plant virus as computed tomography imaging contrast agent

Near-infrared light-responsive nanomaterials in cancer therapeutics

Gold nanorods: their potential for photothermal therapeutics and drug delivery, tempered by the complexity of their biological interactions

Photothermal therapy and photoacoustic imaging via nanotheranostics in fighting cancer

Site-Selective Nucleation and Size Control of Gold Nanoparticle Photothermal Antennae on the Pore Structures of a Virus

Glycantargeted virus-like nanoparticles for photodynamic therapy

Multifunctionalized biocatalytic P22 nanoreactor for combinatory treatment of ER+ breast cancer

Polydopamine-decorated tobacco mosaic virus for photoacoustic/magnetic resonance bimodal imaging and photothermal cancer therapy

Iron/iron oxide core/shell nanoparticles for magnetic targeting MRI and near-infrared photothermal therapy

Comparison study of gold nanohexapods, nanorods, and nanocages for photothermal cancer treatment

First demonstration of gold nanorods-mediated photodynamic therapeutic destruction of tumors via near infra-red light activation

Copper Selenide Nanocrystals for Photothermal Therapy

Biomineralization synthesis of HBc-CuS nanoparticles for near-infrared light-guided photothermal therapy

Improved Stable Indocyanine Green (ICG)-Mediated Cancer Optotheranostics with Naturalized Hepatitis B Core Particles

Construction of Multifunctional Fe3O4-MTX@HBc Nanoparticles for MR Imaging and Photothermal Therapy/Chemotherapy

Biomineralization-Inspired Synthesis of Copper Sulfide-Ferritin Nanocages as Cancer Theranostics

Intracellular Biodegradation of Ag Nanoparticles, Storage in Ferritin, and Protection by a Au Shell for Enhanced Photothermal Therapy

Refactored M13 Bacteriophage as a Platform for Tumor Cell Imaging and Drug Delivery

A Phase II Clinical Trial to Evaluate the Recombinant Vaccine for COVID-19 (Adenovirus Vector) -Full Text View -ClinicalTrials.gov

A Phase 2/3 Study to Determine the Efficacy, Safety and Immunogenicity of the Candidate Coronavirus Disease (COVID-19) Vaccine ChAdOx1 nCoV-19, clinicaltrials.gov

Development of virus-like particles for diagnostic and prophylactic biomedical applications

Bio inspired shielding strategies for nanoparticle drug delivery applications

Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles

Nanoparticle PEGylation for imaging and therapy

Antibody Response against Cowpea Mosaic Viral Nanoparticles Improves In Situ Vaccine Efficacy in Ovarian Cancer

Interaction of Viral Capsid-Derived Virus-Like Particles (VLPs) with the Innate Immune System, Vaccines (Basel)

Horizontal gene transfer events reshape the global landscape of arm race between viruses and homo sapiens

Sourabh Shukla is thanked for help with the graphics shown in Figure 6 .

There are no competing interests to declare.