Viral protein-based nanoparticles (part 2): Pharmaceutical applications

European Journal of Pharmaceutical Sciences 189 (2023) 106558 Available online 9 August 2023 0928-0987/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Viral protein-based nanoparticles (part 2): Pharmaceutical applications Rocio Mellid-Carballal a , b , Sara Gutierrez-Gutierrez a , b , Carmen Rivas a , c , d , Marcos Garcia- Fuentes a , b , c , * a CiMUS Research Center, Universidad de Santiago de Compostela, Spain b Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Universidad de Santiago de Compostela, Spain c Health Research Institute of Santiago de Compostela (IDIS), Universidad de Santiago de Compostela, Spain d Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología (CNB)-CSIC, Spain ARTICLE INFO Keywords: Virus-like particles Virosomes Vaccines Immunostimulation Drug delivery Diagnostics ABSTRACT Viral protein nanoparticles (ViP NPs) such as virus-like particles and virosomes are structures halfway between viruses and synthetic nanoparticles. The biological nature of ViP NPs endows them with the biocompatibility, biodegradability, and functional properties that many synthetic nanoparticles lack. At the same time, the absence of a viral genome avoids the safety concerns of viruses. Such characteristics of ViP NPs offer a myriad of opportunities for theirapplication at several points across disease development: from prophylaxis to diagnosis and treatment. ViP NPs present remarkable immunostimulant properties, and thus the vaccination field has benefited the most from these platforms capable of overcoming the limitations of both traditional and subunit vaccines. This was reflected in the marketing authorization of several VLP- and virosome-based vaccines. Besides, ViP NPs inherit the ability of viruses to deliver their cargo to target cells. Because of that, ViP NPs are promising candidates as vectors for drug and gene delivery, and for diagnostic applications. In this review, we analyze the pharmaceutical applications of ViP NPs, describing the products that are commercially available or under clinical evaluation, but also the advances that scientists are making toward the implementation of ViP NPs in other areas of major pharmaceutical interest. 1. Introduction In the 1960s, an unknown antigen was detected in the serum of an Australian individual during a study of hemophilia patients. Although it was initially linked to the development of leukemia, it was eventually connected to an infection with the hepatitis B virus (HBV). Electron microscopy and immunofluorescence techniques finally revealed that the so-called “Australian antigen” was actually a surface antigen of the HBV, which formed structures similar to viral capsids, but smaller and devoid of genetic material (Blumberg and Alter, 1965; Blumberg, 1977). This finding is considered the first evidence of the existence of virus-like particles (VLPs) (Yan et al., 2015). Since then, many other VLPs have been identified and synthesized. VLPs are defined as supramolecular assemblies of proteins that mimic viral structure but lack genetic material, so they are non-infective (Jennings and Bachmann, 2008). VLPs are either prepared by expressing complete viruses and removing the viral genome, or by direct expression of viral capsid proteins from a vector. They are usually rod-shaped or icosahedral and their size varies between 20 and 200 nanometers in diameter (Jennings and Bachmann, 2008; Dai et al., 2018). Depending on the parental virus, VLPs can be enveloped or not. Non-enveloped VLPs can be simple viral capsids formed by one or two major proteins, such as human papillomavirus (HPV)-VLPs, or complex viral capsids made up of several protein layers, such as rotavirus VLPs (Shirbaghaee and Bolhassani, 2016; Fuenmayor et al., 2017). In enveloped VLPs, such as human immunodeficiency virus-1 (HIV-1)-VLPs, the viral capsid is surrounded by a lipid bilayer from the host cell membrane containing glycoprotein spikes (Fuenmayor et al., 2017). When comparing VLPs to synthetic nanoparticles, the latter are easier to produce since they do not require expression systems and do not need complex downstream processing (Plummer and Manchester, 2011). However, alternatives such as the production of VLPs in edible plants may overcome this limitation (Biemelt et al., 2003). Regarding immunogenicity, VLPs typically exhibit greater immune stimulation compared to synthetic nanoparticles. This result is undesirable when VLPs are intended as delivery vehicles, but strategies have been developed to make “stealth” VLPs (Chen et al., 2016). Finally, although * Corresponding author. E-mail address: [email protected] (M. Garcia-Fuentes). Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps https://doi.org/10.1016/j.ejps.2023.106558 Received 17 February 2023; Received in revised form 10 July 2023; Accepted 4 August 2023 European Journal of Pharmaceutical Sciences 189 (2023) 106558 2 synthetic nanoparticles can be as safe as VLPs, their in vivo elimination is generally less favorable (Plummer and Manchester, 2011). A decade after the discovery of VLPs, Almeida et al. succeeded at including hemagglutinin (HA) and neuraminidase (NA) in a liposome, creating what they called a virosome (Almeida et al., 1975). Virosomes are lipid/viral protein nanometric structures with spherical shape and a diameter of around 60 to 200 nanometers (van der Velden et al., 2022). They are prepared by solubilizing native enveloped viruses, removing the nucleocapsid, to finish with the reconstitution of the viral envelope proteins into a liposome (Fig. 1) (Huckriede et al., 2003; Asadi and Gholami, 2021; Mak and Saunders, 2006). While virosomes share a similar structure with liposomes, their distinct viral glycoprotein coating sets them apart significantly in terms of functionality. The incorporation of antigens into liposomes is challenging and, when achieved, adjuvants are required to elicit strong immune responses (Kersten and Crommelin, 2003). Both requirements are avoided when developing a virosomal vaccine. Moreover, virosomes have a big advantage when endosomal escape is needed (see Section 3.1). Both VLPs and virosomes are highly versatile, where active/functional agents can be incorporated either in their coating layers or in their inner cavity (Fig. 1). Examples of such agents are ligands, drugs, antigenic sequences, contrast agents, and nucleic acids (van der Velden et al., 2022; Asadi and Gholami, 2021; Koudelka et al., 2015; Diaz et al., 2018; Ding et al., 2018). A powerful strategy is to encapsulate the therapeutic agent inside the viral nanoparticle cavity, where it is protected from degradation, while its surface is functionalized with a targeting moiety for a specific cell type (Thong et al., 2019). Other strategies like the attachment of polymers to the surface of VLPs or virosomes can avoid undesirable immunogenicity (Farkas et al., 2013). Besides, these viral protein nanoparticles (ViP NPs; i.e., a term we use to refer collectively to VLPs and virosomes) show lower toxicity and less safety concerns than other biological particles (Yan et al., 2015; Asadi and Gholami, 2021). They can be administered through a variety of parenteral routes, and the possibilities offered by their transmucosal administration are currently being investigated (Mohsen et al., 2020; De Bernardis et al., 2015; Huang et al., 2005; Sedlik et al., 1999). Once in the circulation, ViP NPs are completely degraded in a matter of days (Kaiser et al., 2007). In a previous review paper, we have comprehensively covered these structural, toxicological, and biopharmaceutical issues. We also analyzed the manufacturing process and the formulation strategies (Mellid-Carballal et al., 2023). In this comprehensive review, we focus on medical applications of ViP NPs and the functional characteristics that confer them competitive advantages. Specifically, we delve into the utilization of VLPs and virosomes in the fields of immunology and immunotherapy, shedding light on their clinical use and emerging applications. Furthermore, we analyze ongoing research endeavors investigating the potential use of ViP NPs in other domains, including drug/gene delivery and diagnostics. Finally, we address pivotal mechanistic inquiries, such as the intrinsic immunogenic properties of ViP NPs, their targeting and intracellular trafficking capabilities, and the effective loading of contrast agents within these biomedical platforms. 2. ViP NPs for vaccination Until the 80s, vaccines were based on inactivated or attenuated viruses (Mohsen et al., 2017). Although they showed excellent immunogenicity, safety issues and difficulties in mass production limited their use (Mohsen et al., 2017; Mohsen et al., 2020). The discovery in the 70s that a single viral protein could be enough to induce immunostimulation (Rold˜ ao et al., 2010) laid the foundations for the development of subunit vaccines, which are safer but less immunogenic than conventional vaccines. Nucleic acid vaccines are easier to produce than conventional and subunit vaccines. However, the requirement for an optimal delivery system limited their development (Liu, 2019; Ho et al., 2021). Some DNA vaccines are available in the veterinary field (Chang et al., 2007; Salonius et al., 2007; McLean and Lobetti, 2015), but none are for human use (Ho et al., 2021). mRNA vaccines are a new technology that has demonstrated great value when rapid development is mandatory, as occurred with the COVID-19 pandemic (Dolgin, 2021). Nanoparticle-based vaccines induce excellent immune responses due to their polyvalent surfaces (Bachmann and Jennings, 2010; Zabel et al., 2013). Their nanometric size allows them to freely drain to the lymph nodes (Fifis et al., 2004), where they are taken up by antigen-presenting cells (APCs) (Manolova et al., 2008). Although nanoparticles have an adjuvant effect by themselves, they can also be combined or conjugated List of abbreviations AIT Allergen-specific immunotherapy APC Antigen-presenting cell CCMV Cowpea chlorotic mottle virus CED Convection-enhanced delivery CpG Cytosine-phosphate-guanosine oligodeoxynucleotides CSP Circumsporozoite protein CT Computed tomography CTL Cytotoxic T lymphocyte DODAC N,N-dioleyl-N,N-dimethylammonium chloride DOSPER 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide DOTAP 1,2-dioleoyl-3-trimethylammonium propane DOX Doxorubicin EMA European Medicines Agency EMHV Erythro-magneto-hemagglutinin virosome FDA Food and Drug Administration HA Hemagglutinin HBcAg Hepatitis B core antigen HBsAg Hepatitis B surface antigen HBV Hepatitis B virus HEV Hepatitis E virus HIV Human immunodeficiency virus HPV Human papillomavirus IRIV Immunopotentiating reconstituted influenza virosome MERS-CoV Middle East respiratory syndrome coronavirus MHC Major histocompatibility complex miRNA micro-RNA MPL 3-O-desacyl-4 ′ -monophosphoryl lipid A MRI Magnetic resonance imaging NA Neuraminidase NIR Near-infrared PEG Polyethyleneglycol PET Positron emission tomography RBD Receptor-binding domain SARS-CoV Severe acute respiratory syndrome coronavirus siRNA Small interference RNA SV40 Simian virus 40 TAA Tumor-associated antigen TAT Transactivator of transcription TLR Toll-like receptor TMV Tobacco mosaic virus tPA Tissue plasminogen activator ViP NP Viral protein nanoparticle VLP Virus-like particle WHO World Health Organization R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 3 to other adjuvants to increase their immunogenicity. Moreover, they are safe and easy to produce (Gomes et al., 2017). Nanoparticle-based vaccines include liposomes, lipid nanoparticles, cationic polymers, viral vectors, and ViP NPs. VLPs and virosomes are the only biological nanoparticle-type antigenic structures that have been approved as vaccines for human use (Gomes et al., 2017). The current diversity of vaccine structures is visually depicted in Fig. 2. 2.1. Immunostimulation mechanisms of ViP NPs 2.1.1. VLPs VLPs display antigens in a similar conformation to the natural virus. The repetitive pattern of antigens on the VLP surface (multivalent presentation) enhances the activation of B cells by crosslinking multiple B- cell receptors. Therefore, VLPs induce stronger humoral responses than subunit vaccines (Shirbaghaee and Bolhassani, 2016; Donaldson et al., 2018), even in the absence of adjuvants (Yan et al., 2015; Mohsen et al., 2017; Mohsen et al., 2020; Donaldson et al., 2018; Jeong and Seong, 2017). In addition, their size range is optimal for draining to the lymph nodes, where they can be efficiently taken up by dendritic cells (Yan et al., 2015; Mohsen et al., 2017; Jeong and Seong, 2017). Internalized VLPs are processed and presented by major histocompatibility complex (MHC) class II molecules for the activation of T helper cells (Fig. 3). Due to their highly ordered structure, VLPs resemble pathogenassociated molecular patterns, so they are recognized by specific receptors, triggering an interferon response and inflammation, and increasing MHC class I antigen presentation and, consequently, the priming of cytotoxic T lymphocytes (CTLs) (Fig. 3) (Yan et al., 2015; Shirbaghaee and Bolhassani, 2016). As VLPs can induce both humoral and cellular responses, they are of great interest for vaccination strategies, with prophylactic and therapeutic objectives. The strong immunogenicity of VLPs, together with their self-adjuvant properties and their high stability, results in stronger immune responses with lower doses applied compared to subunit vaccines, leading to cost reduction (Koudelka et al., 2015; Mohsen et al., 2020; Rold˜ ao et al., 2010; Shirbaghaee and Bolhassani, 2016). Finally, VLPs can be engineered to present two or more different antigens (chimeric VLPs) (Koudelka et al., 2015). This is particularly useful when intending to develop multivalent vaccines or to present an antigen with no self-assembling properties in the immunostimulant structural context of a VLP. Environmental exposure to prevalent strains of a VLP parent virus, or previous vaccination with a VLP vaccine may generate anti-VLP antibodies, thus having deleterious interference (Jegerlehner et al., 2010). This can be overcome by alternating between different VLP vectors (Zhou et al., 2011), or by masking VLP surface antigens (Jariyapong et al., 2013). 2.1.2. Virosomes The paradigmatic example of virosomes are influenza virosomes (Crisci et al., 15), which allow influenza antigens to be presented in a non-pyrogenic yet immunogenic platform (Almeida et al., 1975; Metcalfe and Gluck, 2004). The two glycoproteins of the influenza virus membrane, HA and NA, permit virosomes to fuse with the plasma Fig. 1. Synthesis and modification strategies of ViP NPs. (A) Production of VLPs using cell-based or cell-free expression systems, and subsequent modification through genetic engineering, chemical bioconjugation, non-covalent surface assembly, or by cargo encapsulation. HPQ: homophenylalaninylmethane sequence of a biotin-mimicking peptide. (B) Main steps in the production of virosomes from enveloped viruses, and potential modifications, including integration in the lipid membrane, chemical coupling, and encapsulation. Created with BioRender.com. R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 4 membrane of non-phagocytic cells. There, the antigen is processed and presented on MHC class I molecules that induce a specific CTL response. HA also targets virosomes to receptors containing sialic acid on the membranes of APCs, promoting their endocytosis. After virosome degradation in the endosome, the antigen is presented on MHC class II molecules and induces a humoral response (Fig. 3) (Mak and Saunders, 2006). The capacity of influenza virosomes to induce strong humoral and cellular immune responses makes them useful as influenza vaccines. Moreover, they show intrinsic adjuvant capacity, enhancing the immune Fig. 2. Types of vaccines. The structural differences among the diverse particles used for vaccination are represented. Created with BioRender.com. Fig. 3. Immunostimulation by VLPs and virosomes. Both ViP NPs are taken up by APCs, such as dendritic cells, where antigens are presented on class I and II MHC molecules. Therefore, they activate CD4+and CD8+T cells that eventually induce humoral and cellular immune responses, respectively. Created with Bio- Render.com. R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 5 response to unrelated antigens. When a substantial amount of external lipids is added to influenza virosomes, the resulting platforms are termed immunopotentiating reconstituted influenza virosomes (IRIVs). IRIVs are biodegradable and biocompatible, showing an excellent safety profile. They can be administered by most routes, combined with other adjuvants, and used for multiantigen delivery (Asadi and Gholami, 2021). These properties have resulted in the FDA authorization of IRIV-based nanocarriers for human use. Their major limitation is their rapid disintegration in the bloodstream (Asadi and Gholami, 2021; Liu et al., 2015), although it can be overcome by conjugating a targeting ligand that drives virosomes to their target sites within a short time after administration. The applications of virosomes are not limited to viral infections. For example, they can deliver tumor-associated antigens (TAAs), serving simultaneously as carriers and adjuvants, and promoting the generation of immune responses against cancer cells (Wiedermann et al., 2010). 2.2. ViP NPs in prophylactic vaccination The immunogenic properties of VLPs and virosomes have lead to the approval of several vaccines for the prevention of infectious diseases. Also, some candidates are undergoing clinical trials and many prototypes are under preclinical investigation. Here, we will focus on VLP- based and virosome-based vaccines that are commercially available or have reached clinical evaluation, which are summarized in Tables 1 and 2, respectively. 2.2.1. Hepatitis B virus In the 1980s, hepatitis B surface antigen (HBsAg)-VLPs isolated from the blood of infected individuals were commercialized as vaccines under the trade names Heptavax B® and Hevac B® (Mohsen et al., 2017; Gomes et al., 2017; Tornesello et al., 2022). Although they were considered the first VLP-based vaccines, they were irregular lipoprotein complexes, different from the highly ordered protein structures generally described as VLPs (Jennings and Bachmann, 2008). The safety issues related to blood products, the dependence on blood availability and the advances in recombinant DNA technology and biotechnology led to the development of recombinant HBsAg (Rold˜ ao et al., 2010; Gomes et al., 2017). Second-generation vaccines Recombivax HB® (Merck and Co., Inc.) and Engerix B® (GlaxoSmithKline S.A.) were approved by the FDA in 1986 and 1989, respectively. Both vaccines were based on the recombinant expression of HBsAg in Saccharomyces cerevisiae, where HBsAg spontaneously assembles into 20 nm lipid-containing particles. They were safer and more immunogenic than first-generation vaccines (Jennings and Bachmann, 2008; Shirbaghaee and Bolhassani, 2016; Diaz et al., 2018; Mohsen et al., 2017; Rold˜ ao et al., 2010; Donaldson et al., 2018; Tornesello et al., 2022). Even though the commercialization of these vaccines meant a great success, their combination with adjuvants could improve responder rates in immunocompromised subjects and increase protection without increasing the number of doses (Jennings and Bachmann, 2008). Fendrix® vaccine, adjuvanted by AS04, received marketing authorization from the EMA in 2005 to be used in patients with renal insufficiency (Machiels et al., 2019). More recently, the third generation of HBV-VLP-based vaccines was developed to further increase protection levels, particularly in some sectors of the population, such as immunocompromised patients Table 1 ViP NP-based prophylactic vaccines that have received marketing authorization. Name Target agent Antigen ViP NP type Expression system Manufacturer First approval Heptavax B® Hepatitis B virus HBsAg VLP Blood-derived Merck Pharmaceuticals 1981 (FDA) Discontinued in 1990 Hevac B® Hepatitis B virus HBsAg VLP Blood-derived Institute Pasteur 1981 (France) Recombivax HB® Hepatitis B virus HBsAg VLP Saccharomyces cerevisiae Merck and Co., Inc. 1986 (Germany and FDA) Engerix B® Hepatitis B virus HBsAg VLP Saccharomyces cerevisiae GlaxoSmithKline S.A. 1986 (Belgium) 1989 (FDA) Fendrix® Hepatitis B virus HBsAg VLP Saccharomyces cerevisiae GlaxoSmithKline S.A. 2005 (EMA) Sci-B-Vac® Hepatitis B virus S, Pre-S1, and Pre-S2; M and L proteins VLP Chinese hamster ovary cells VBI Vaccines (Delaware) Inc. 2017 (Israel and East Asia) GenHevac B® Hepatitis B virus S, Pre-S1, and Pre-S2; M protein VLP Hansenula polymorpha Pasteur-M´ erieux Aventis 1989 (France) Heplisav B® Hepatitis B virus HBsAg VLP Hansenula polymorpha Dynavax Technologies Corporation 2017 (FDA) Gardasil® Human papillomavirus L1 protein (HPV 6, 11, 16, and 18) VLP Saccharomyces cerevisiae Merck and Co., Inc. 2006 (FDA) Cervarix® Human papillomavirus L1 protein (HPV 16, and 18) VLP Trichoplusia ni (High Five) cells GlaxoSmithLine S.A. 2009 (FDA) Gardasil® 9 Human papillomavirus L1 protein (HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58) VLP Saccharomyces cerevisiae Merck Sharp & Dohme Corp. 2017 (FDA) Cecolin® Human papillomavirus L1 protein (HPV 16, and 18) VLP E. coli Xiamen Innovax Biotech Co., Ltd. 2019 (China) Hecolin® Hepatitis E virus L1 protein (HPV 6, 11, 16, and 18) VLP E. coli Xiamen Innovax Biotech Co., Ltd. 2011 (China) Mosquirix® Plasmodium falciparum Secreted circumsporozoite protein (CSP) HBsAg- VLPs Saccharomyces cerevisiae GlaxoSmithKline S.A. 2022 (WHO prequalification) Epaxal® Hepatitis A virus Formalin-inactivated Hepatitis A antigen (strain RG-SB) IRIV HAV virions: MRC-5 cells Berna Biotech Co., Ltd. Crucell 1994 (Switzerland) 1999 (EMA) Discontinued in 2011 Inflexal® V Influenza virus HA and NA from annual strains IRIV Fertilised chicken eggs Berna Biotech Co., Ltd. Crucell 1997 (Switzerland) 2001 (EMA) Discontinued in 2011 Porcilis® PCV Porcine circovirus type 2 ORF2 capsid protein VLP Trichoplusia ni (High Five) cells Intervet International BV 2009 (EMA) Ingelvac CircoFLEX® Porcine circovirus type 2 ORF2 capsid protein VLP Spodoptera frugiperda 9 (Sf9) cells Boehringer Ingelheim Vetmedica GmbH 2008 (EMA) R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 6 Table 2 ViP NP-based prophylactic vaccines that have reached clinical trials. Name Target agent Antigen ViP NP type Expression system Phase Registration number Status Hepatitis B virus HBsAg VLP Transgenic tubers (Solanum tuberosum) Hepatitis B virus HBsAg VLP Transgenic lettuce (Lactuca sativa L.) R21/Matrix-M Malaria CPS from Plasmodium falciparum HBsAg-VLP III NCT04704830 Active, not recruiting II NCT05252845 Recruiting MalariVax (ICC-1132) Malaria CPS from Plasmodium falciparum HBcAg-VLP E. coli I NCT00587249 Completed PEV301&302 Malaria Synthetic peptides derived from apical membrane antigen-1 (PEV301) and from CSP (PEV302) from Plasmodium falciparum Virosome Ib NCT00513669 Completed ACAM FLU-A Influenza virus M2 peptides from the influenza A virus HBcAg-VLP E. coli I NCT00819013 Completed Influenza virus VLP IIa NCT00903552 Completed NanoFLu (Quad-NIV) Influenza virus Recombinant HA protein VLP Sf9 insect cells III NCT04120194 Completed Norwalk virus Major capsid protein (VP1) VLP Sf9 insect cells Norwalk virus Major capsid protein (VP1) VLP Transgenic potatoes Norwalk virus VP1 VLP Baculovirus expression system I NCT00806962 Completed VAI-VP705 Human parvovirus B19 VP1 and VP2 proteins VLP baculovirusinsect cell expression system I/II NCT00379938 Terminated VRC – CHKVLP059–00- VP Chikungunya Virus E1, E2, and capsid proteins VLP VRC293 cells II NCT02562482 Completed HIV p17/p24:Ty-VLP HIV p17 or p24 Ty VLP S. cerevisiae I II NCT00001053 Completed CoVLP SARS-CoV-2 Mutated S2P (stabilized prefusion form) SARS-CoV-2 VLP Nicotiana benthamiana I NCT0445000 Complete I/II NCT05065619 Terminated (for ethical reasons to prioritize ancentral vaccination) II/III NCT04636697 Active, not recruiting III NCT05040789 Withdrawn (further development terminated) VBI-2902 VBI-2905 SARS-CoV-2 B.1.351 variant S protein (prefusion state) Enveloped MLVVLP I NCT04773665 Completed VBI-2901 SARS-CoV-2 SARS-CoV MERS S protein Enveloped MLVVLP I NCT05548439 Active, not recruiting ABNCoV2 SARS-CoV-2 RBD from S protein Acinetobacter phage AP205 capsid-like particle E. coli (Tag-CLP) Drosophila melanogaster (RBD-Catcher) I NCT04839146 Completed II NCT05077267 Active, not recruiting III NCT05329220 Active, not recruiting SARS-CoV-2 VLP vaccine SARS-CoV-2 S, E, M, and N proteins VLP I NCT0481828 Completed II NCT04962893 Completed RBD SARS-CoV-2 HBsAg VLP Vaccine SARS-CoV-2 RBD HBsAg-VLP Yeast I/II ACTRN12620001308987 Stopped early est vaccine no longer developed for commercial reasons) I/II ACTRN12620000817943 Recruiting LYB001 SARS-CoV-2 RBD VLP vector II/III NCT05602558 Not yet recruiting II NCT05663086 Not yet recruiting II/III NCT05137444 Not yet recruiting R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 7 (Shirbaghaee and Bolhassani, 2016; Diaz et al., 2018; Donaldson et al., 2018). Sci-B-Vac®, developed by VBI Vaccines Inc., was first approved in Israel and East Asia in 2017 and is composed of three surface antigens (S, Pre-S1, and Pre-S2), together with M and L proteins, expressed in mammalian Chinese hamster ovary cells (Mohsen et al., 2017; Rold˜ ao et al., 2010). It combines glycosylated and non-glycosylated HBsAg, thus eliciting higher immunogenicity than yeast-derived vaccines. The Gen- Hevac B® vaccine contains the same three surface antigens together with the M protein (Rold˜ ao et al., Oct), expressed in Hansenula polymorpha yeast, and is registered in India (Shivananda et al., 2006). Finally, the vaccine Helipsav B, manufactured by Dynavax Technologies Corporation and approved in the United States, contains HBsAg-VLPs adjuvanted with CpG sequence 1018 (Tornesello et al., 2022). A main goal in vaccine development is the production of edible vaccines. Current studies take advantage of the assembly of HBV-VLPs in transgenic plants (Jennings and Bachmann, 2008; Fuenmayor et al., 25). Thanavala et al. generated HBsAg transgenic tubers in Solanum tuberosum. Subjects who have previously received a vaccine against hepatitis B showed higher titers of specific anti-HBsAg antibodies after eating the transgenic potatoes (Thanavala et al., 2005). Kapusta et al. introduced the HBsAg gene into Agrobacterium tumefaciens to obtain transgenic lupin and lettuce. Human volunteers fed with this transgenic lettuce developed a specific humoral response (Kapusta et al., 2001). 2.2.2. Human Papillomavirus In the 1990s, it was discovered that the L1 major capsid of HPV can self-assemble into VLPs. They were identical to the wild-type virus and induced both humoral and cellular responses (Jennings and Bachmann, 2008; Fuenmayor et al., 25; Rold˜ ao et al., 2010; Tornesello et al., 2022; Roy and Noad, 2008). Currently, three HPV VLP-based vaccines are available in Europe and the United States: Gardasil® (Merck and Co., Inc.), Cervarix® (GlaxoSmithKline S.A.), and Gardasil® 9 (Shirbaghaee and Bolhassani, 2016; Diaz et al., 2018; Donaldson et al., 2018). Gardasil®, approved by the FDA in 2006, is a tetravalent vaccine composed of VLPs of the recombinant L1 protein of HPV types 6, 11, 16, and 18. VLPs of each HPV type are produced in a separate culture of Saccharomyces cerevisiae, adsorbed onto aluminum hydroxyphosphate sulfate, combined, and formulated for intramuscular administration (Jennings and Bachmann, 2008; Fuenmayor et al., 25; Mohsen et al., 2017; Rold˜ ao et al., 2010; Tornesello et al., 2022). Cervarix®, licensed in Europe in 2007, is a bivalent vaccine against HPV types 16 and 18. In this case, a recombinant baculovirus containing the L1 protein was used to infect Trichoplusia ni insect cells. The purified L1 protein is assembled into VLPs, which are adjuvanted with AS04 (Jennings and Bachmann, 2008; Fuenmayor et al., 2017; Mohsen et al., 2017; Rold˜ ao et al., 2010; Tornesello et al., 2022). Gardasil® 9, licensed in the USA in 2014, is a variation of Gardasil® that covers nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) (Mohsen et al., 2017; Tornesello et al., 2022). Gardasil® and Cervarix® are still in clinical trials in some countries (Rold˜ ao et al., 2010). The results show that they induce not only protection against the HPV serotypes included in the vaccine, but also some response to others not included (Smith et al., 2007; Harari et al., 2016). Another clinical study is comparing the immunogenicity of fractional doses (one-fifth of the standard dose) of Cervarix® and Gardasil® 9 (NCT04235257) (Tornesello et al., 2022). The main limitation of commercialized vaccines is their high production costs (Tornesello et al., 2022), which most developing countries cannot afford. Xiamen Innovax Biotech Co., Ltd. (Innovax) has recently developed a bivalent VLP vaccine against HPV types 16 and 18 using an Escherichia coli expression system (Qian et al., 2020). Clinical trials show excellent safety and similar induction of neutralizing antibodies to the reference vaccines (Gu et al., 2017; Qiao et al., 2020). It has been first marketed in China in 2019 under the name Cecolin®. Gardasil®, Cervarix®, Gardasil® 9, and Cecolin® have received WHO prequalification, which ensures they meet international quality, safety, and efficacy standards, and accelerates their introduction to the global public vaccine market (Regan and Graham, 2021). Additional clinical studies would help to expand vaccine use, decrease costs, and address supply needs (Kuter et al., 2021). Some studies are looking for a therapeutic vaccine against cervical cancers related to HPV (Chen et al., 2011; Cai et al., 2022). In addition, the production of HPV VLPs in transgenic plants (potatoes) has been achieved by two groups (Biemelt et al., 2003; Garcea and Gissmann, 2004; Warzecha et al., 2003; Naupu et al., 2020). This would open the possibility of developing edible vaccines, which would be very beneficial for patient comfort. 2.2.3. Hepatitis E virus Innovax has developed HEV 293, a VLP-based vaccine produced in E. coli using a truncated form of the viral capsid protein of the hepatitis E virus (HEV). It showed both high immunogenicity and safety after intramuscular administration in clinical trials (Zhu et al., 2010) and was approved in China in 2011 under the trade name Hecolin® (Fuenmayor et al., 2017; Gomes et al., 2017; Donaldson et al., 2018; Roy and Noad, 2008). 2.2.4. Malaria GlaxoSmithKline developed the first advanced vaccine against the pre-erythrocytic stage of Plasmodium falciparum (Hawkes, 2015). RTS, S/AS01 vaccine (Mosquirix®) is produced by fusing parts of the secreted circumsporozoite protein (CSP) of the parasite to the surface of HBV VLPs. It was produced in yeast and formulated with AS01 adjuvant. Phase III clinical trials showed that three monthly intramuscular administrations of Mosquirix® to children from 6 weeks to 17 months old help to protect them against malaria (RTS SCT Partnership, 2012; RTS SCT Partnership, 2015). Although it is still not approved by FDA and EMA, the latter issued a positive opinion for its use outside the European Union (European Medicines Agency, 2015). Currently, Mosquirix® is administered to populations where malaria is prevalent. At the same time, several clinical trials evaluating Mosquirix® are still ongoing. In September 2022, it has been prequalified by the WHO, being the first case for a malaria vaccine (GSK, 2022). A VLP-based vaccine, named R21, showed positive results in phase I/ IIa clinical trials. In contrast to Mosquirix®, here the CPS antigen is directly fused to HBsAg, so the ratio CSP/VLP is higher. Moreover, it is formulated with potent adjuvants (Matrix-M and Abisto-100) (Plummer and Manchester, 2011). A phase I/IIb clinical trial in children in Burkina Faso (NCT03896724) suggests that vaccination with R21/Matrix-M could have a significant impact in areas of highly seasonal malaria transmission in Africa (Cornuz et al., 2008). This trial has progressed to phase III (NCT04704830). To assess the safety and immunogenicity of R21/Matrix-M in Asian populations, a phase II clinical trial in Thai adults was planned in February 2022 (NCT05252845). MalariVax (ICC-1132) is a candidate vaccine produced in E. coli where CSP is fused to the surface of hepatitis B core antigen (HBcAg)- VLPs (Nauck et al., 2021). The results of a phase I trial demonstrated clinical efficacy against malaria (Torresi et al., 2011). Finally, there are some virosome formulations for malaria vaccination under clinical evaluation. PEV301 and PEV302 are virosomal formulations containing synthetic peptides derived from apical membrane antigen-1 and CSP from Plasmodium falciparum, respectively. Vaccination of healthy semi-immune adults and children with a combination of PEV301 and PEV302 produced a significant decrease in malaria incidence without important adverse effects (Passalacqua et al., 2018). 2.2.5. Hepatitis A virus Epaxal®, commercialized in 1994 (Herzog, 2006) consists of formalin-inactivated hepatitis A antigen adsorbed to the surface of IRIVs (Bovier, 2008). The substitution of aluminum-based adjuvants by IRIVs resulted in similar immunogenicity but fewer local reactions (Holzer et al., 1996). After a single injection, Epaxal® is highly immunogenic R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 8 and well-tolerated (Bovier, 2008). The formulation for children, called HAVpur® Junior or Epaxal® Junior, has shown great potential for mass vaccination programs (Van Der Wielen et al., 2007). A phase IV clinical trial in India reported high immunogenicity and safety (Jain et al., 2015). 2.2.6. Influenza virus A virosome-based vaccine against influenza was first marketed in Switzerland in 1997 under the name Inflexal® V. Currently, it is approved in many countries, in some of them with a different name: InfectoVac Flu® in Germany, Isiflu® V in Italy, and Viroflu® in the United Kingdom (Herzog et al., 2009). It is a trivalent vaccine that results from the mixture of three monovalent virosome pools, each of them carrying the specific HA and NA from the influenza strains annually recommended by the WHO and the EMA (Mischler and Metcalfe, 2002; Gerdil, 2003). This is the only adjuvanted influenza vaccine that has been licensed for all age groups, showing excellent tolerability and high immunogenicity, not only in healthy adults but also in immunocompromised individuals (Herzog et al., 2009). Alternative targets to HA and NA, such as the influenza M2 gene with a low mutation rate eliminate the requirement for annual evaluations of influenza vaccines (Kaiser, 2006). Although M2 does not self-assemble into VLPs, it can be displayed on the surface of recombinant VLPs to increase its immunogenicity (Neirynck et al., 1999). Sanofi Pasteur developed a VLP vaccine based on HBcAg-VLPs produced in E. coli and decorated with M2 peptides from the influenza A virus on their surface (Rold˜ ao et al., 2010). Phase I clinical trials show that this vaccine, ACAM FLU-A, induces seroconversion to M2 ectodomain antigens, especially when adjuvanted with Stimulon QS 21 (Fiers et al., 2009). The pharmaceutical company Novavax developed a trivalent vaccine based on influenza VLPs, including the following strains: H1N1 A/ Brisbane/59/2007, H3N2 A/Brisbane/10/2007, and B/Florida/04/ 2006. They reported positive results in preclinical and clinical trials (Rold˜ ao et al., 2010). Novavax also developed a quadrivalent influenza vaccine, called NanoFLu, produced in the baculovirus-insect cell expression system and adjuvanted with Novavax’s patented saponin-based Matrix-M™. Phase 3 clinical trials comparing NanoFLu and Fluzone, an inactivated tetravalent influenza vaccine, demonstrated enhanced humoral and cellular immune responses with NanoFLu and a similar safety profile (Shinde et al., 2022). 2.2.7. Norwalk virus The main capsid protein of the Norwalk virus (VP1) undergoes spontaneous assembly into VLPs when expressed in Spodoptera frugiperda 9 (Sf9) insect cells. NV-VLPs have also been produced in transgenic plants (potatoes, tomatoes) (Jennings and Bachmann, 2008; Fuenmayor et al., 2017). In phase I clinical trials, oral administration of NV-VLPs, either in water or in transgenic potatoes, induced mucosal IgA production (Ball et al., 1999; Tacket et al., 2000; Tacket et al., 2003). LigoCyte Pharmaceuticals Inc. is developing two formulations of NV- VLPs as vaccine candidates (Rold˜ ao et al., 2010). One of them is a dry powder composed of lyophilized VLPs combined with chitosan and GlaxoSmithKline’s MPL adjuvant. Phase I clinical studies showed good tolerance and high immunogenicity after intranasal administration (El-Kamary et al., 2010). The other one is a liquid containing VLPs, alum, and MPL for intramuscular administration (WO2013009849A1). 2.2.8. Human parvovirus VLPs composed of VP1 and VP2 proteins of human parvovirus B19 can be produced in a baculovirus-insect cell expression system (Kajigaya et al., 1991). A phase I/II clinical trial in combination with MF59 adjuvant had to be halted because of the development of rashes in some participants (Shira et al., 2011). Due to the possible relation between this adverse effect and the phospholipase A2 activity of the VP1 protein, a parvovirus B19 VLP vaccine lacking phospholipase A2 activity has been produced in human embryonic kidney 293T cells. Results of preclinical trials showed a decrease in inflammatory responses in mice without affecting immunogenicity (Suzuki et al., 2022). Therefore, these mutant B19 VLPs could be good candidates for human vaccination. 2.2.9. Chikungunya virus A phase II clinical study (NCT02562482) evaluated the safety and immunogenicity of a Chikungunya VLP-based vaccine (VRC – CHKVLP059–00-VP) composed of E1, E2, and capsid proteins of Chikungunya virus. The administration of two intramuscular injections separated by a period of 28 days was safe and induced high titers of specific antibodies (Chen et al., 2020). 2.2.10. Human immunodeficiency virus A vaccine candidate was developed by fusing the HIV structural proteins p17 or p24 to Ty VLPs (Adams et al., 1987). A phase I study reported that the vaccine was immunogenic and well tolerated in healthy volunteers (Weber et al., 1995). Despite increasing CD4 counts in asymptomatic seropositive subjects, significant humoral or cellular responses were not achieved in a pilot phase II clinical trial (Peters et al., 1997). 2.2.11. SARS-CoV-2 Vaccines against SARS-CoV-2 target mainly the spike (S) protein, and more specifically, its receptor-binding domain (RBD), which is associated with increased transmissibility of the virus (Prates-Syed et al., 2021; Sharifzadeh et al., 2022). At least six VLP-based vaccines developed against SARS-CoV-2 have reached clinical trials. Medicago developed the CoVLP, displaying the stabilized prefusion form of the S protein, and adjuvanted with AS03 from Glaxo Smith Kline. After the administration of two doses, volunteers showed higher neutralizing antibody titers in serum than convalescent individuals (Lehner et al., 1992). CoVLP has reached phase II/III trials (NCT04636697). VBI Vaccines used its proprietary technology for the development of enveloped VLPs, and produced two monovalent VLP-based vaccines containing the S protein in its prefusion state: VBI-2902, directed against the ancestral/Wuhan SARS-CoV-2 strain, and VBI-2905, directed to the beta (B.1.351) variant. Both are adjuvanted with aluminum phosphate, and they have completed phase I trials (NCT04773665). The Radboud University together with AdaptVac developed the ABNCoV2 candidate, consisting of the RBD from the S protein displayed in a bacteriophage scaffold using SpyTag/SpyCatcher technology. Basically, the RBD was genetically fused to the Catcher and expressed in Drosophila melanogaster Schneider 2 insect cell system (ExpreS2). On the other hand, the Tag was added to the capsid protein of Acinetobacter phage AP205 and expressed in E. coli. Finally, both proteins were mixed, resulting in a VLP displaying the RBD. Moreover, during its production in E. coli, the VLP encapsulates bacterial RNA that may serve as a Th1- type adjuvant via Toll-like receptors (TLRs) 7/8 (Fougeroux et al., 2021). The vaccine is currently undergoing phase II/III clinical trials (NCT05077267, NCT05329220). SpyBiotech, in collaboration with the Serum Institute of India (SIIPL), developed another vaccine candidate that uses the SpyTag/ SpyCatcher technology to display the RBD antigen on HBsAg-VLPs. The construct is produced in a yeast expression system. This candidate is in phase I/II trials in Australia (ACTRN12620000817943). One of them (ACTRN12620001308987), has stopped early since the test vaccine was no longer developed for commercial reasons. The Scientific and Technological Research Council of Türkiye (TÜB˙ ITAK) has developed a VLP-based vaccine including the four structural proteins of SARS-CoV-2 (S, E, M, and N proteins). This vaccine has completed phase II trials (NCT04962893) (Mandal, 2021). Yantai Patronus Biotech developed the LYB001 candidate, consisting of the RBD antigen displayed on a VLP vector, which has also reached phase II/III trials (NCT05663086, NCT05602558). Once past the urgent need for vaccine candidates of the COVID-19 R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 9 pandemic, efforts have been directed towards the rational design of versatile platforms that enable rapid responses to new variants. Also, the development of broad-spectrum vaccines against different coronaviruses might help to deal with future pandemics. In this sense, VBI Vaccines has developed a trivalent vaccine (VBI-2901) that uses enveloped VLPs to express the S proteins of SARS-CoV-2, SARS-CoV, and MERS- CoV (Sharifzadeh et al., 2022). After successful results in the preclinical phase, it has entered phase I clinical trials (NCT05548439). Regarding virosome-based vaccines, some candidates contain the RBD antigen from the S protein of SARS-CoV-2 integrated into the lipid membrane of virosomal nanoparticles. One of these candidates is formulated as a thermostable powder vaccine that can be stored at 4 ◦C or even higher temperatures. It is intended for intranasal administration, aiming to induce mucosal immunity in the upper respiratory tract. Up to now, it has shown safety and both humoral and cellular immune responses in rats (Cremona et al., 2022). However, to our knowledge, no virosome-based candidates have entered the clinical phase up to now. 2.2.12. Veterinary infections Although most commercialized vaccines are based on conventional platforms, veterinary vaccinology research is increasingly focusing on subunit vaccines and, especially, in VLPs. In veterinary vaccination, the lower doses of antigen required in VLP vaccines in comparison to monomeric ones is a key factor, since the cost of a vaccine needs to be compared to the value of the animal (Crisci et al., 2012). There are two commercialized VLP-based veterinary vaccines, both against porcine circovirus type 2. The first one, Porcilis PCV, consists of VLPs expressing the ORF2 capsid protein of porcine circovirus type 2 in a baculovirus-insect cell expression system, assembled, and formulated with alfa-tocopherol and a liquid paraffin-based adjuvant (Fan et al., 2007). It was approved for vaccinating pigs at three days of age (Shirbaghaee and Bolhassani, 2016; European Medicines Agency, 2009). The latest one, called Ingelvac CircoFLEX, is based on the same antigen and expression system but adjuvanted with an aqueous polymer (Impran- FLEX). However, it has still not been demonstrated whether this recombinant protein assembles into VLPs (Crisci et al., 2012). Several VLP-based veterinary vaccines are undergoing clinical trials. Some examples are VLPs from parvovirus (Ju et al., 2011), mink enteritis virus (Wu et al., 2020), chicken anemia virus (Tseng et al., 2019), Newcastle disease virus (Park et al., 2014), influenza virus (El-Husseiny et al., 2021), and Bluetongue virus (P´ erez de Diego et al., 2011). In addition, human, plant, or animal VLPs are being studied for the display of animal virus antigens (Crisci et al., 2012). 2.3. ViP NPs in therapeutic vaccination Our immune system has evolved to avoid reactions against selfantigens. However, many chronic diseases are related to the accumulation of self-proteins or the excessive production of inflammatory cytokines (Gomes et al., 2017). In these cases, overcoming self-tolerance would let our immune system fight the disease. Monoclonal antibodies are a successful strategy, but their high costs of production limit their application. Therapeutic vaccination is a cost-effective alternative approach (Gomes et al., 2017). ViP NPs are great candidates as vehicles for the presentation of selfantigens (Yan et al., 2015). The ordered and repetitive antigen presentation allows to overcome B-cell tolerance, thus inducing the production of antibodies to self-proteins implicated in the disease (Yan et al., 2015; Jennings and Bachmann, 2008; Neek et al., 2019; Jeong and Seong, 2017). Up to now, there are no ViP NP-based products in the market for therapeutic vaccination, but great strides have been made in the preclinical and clinical settings. In this section, we will review the ViP NP-based vaccines undergoing clinical evaluation for therapeutic purposes, which are summarized in Table 3. 2.3.1. Cancer Cancer vaccines aim to induce specific CTL responses toward cancer cells. To do so, it is necessary to stimulate the immune system against TAAs (Neek et al., 2019). Therapeutic vaccines may have several advantages over chemotherapy (Koudelka et al., 2015), such as possible outpatient administration, fewer side effects, avoidance of drug resistance, and long-term memory. However, cancer vaccines consisting of Table 3 ViP NP-based therapeutic vaccines currently in clinical trials. Name Application Antigen ViP NP type Phase Registration number Status CYT004-MelQbG10 Malignant melanoma Melan-A/Mart1 Qß-VLP II NCT00651703 Completed IIa NCT00306514 Completed IIa NCT00306566 Completed II NCT00306553 Completed Vidutolimod (CMP- 001) Pancreatic cancer No TAA Qß-VLP Ib/II NCT04387071 Terminated (drug no longer available) Melanoma II/III NCT04695977 Active, not recruiting II NCT04698187 Active, not recruiting Ib NCT03084640 Completed Cervical cancer E7 oncoprotein L1 HPV-VLP PEV7 Recurrent vulvovaginal candidiasis Aspartyl proteinase-2 from Candida albicans Virosome I NCT01067131 Completed CAD106 Alzheimer´s disease Beta-amyloid(1–6) peptide Qβ-VLP IIb NCT01097096 Completed II/III NCT02565511 Terminated CYT006-AngQb Hypertension Modified angiotensin-II peptide Qβ-VLP I/II NCT00500786 Completed CYT002-NicQb Nicotine dependence Nicotine Qβ-VLP II NCT00369616 Completed CYT013-IL1bQb Type II diabetes Interleukin-1β Qβ-VLP I NCT00924105 Completed PEV2A PEV2B Chronic hepatitis C One CD4 epitope and two CD8 epitopes Virosome I NCT00445419 Completed CYT003-QbG10 House dust mite allergy No allergen CYT003-QßG10 VLP II NCT00800332 Completed Allergic bronchial asthma II NCT00890734 Completed CYT005-AllQbG10 House dust mite allergy House dust mite allergen extract CYT003-QßG10 I/II NCT00652223 Completed II NCT00574704 Completed II NCT00574223 Completed CYT005-AllQbG10 Grass pollen allergy Grass pollen allergen CYT003-QßG10 I/II NCT00293904 Completed Qb-Der p 1 Synthetic sequence of allergen Der p 1 Qβ-VLP I Fel-CuMV (HypoCat™) Cat allergy sympthoms Major cat allergen Fel d 1 CMV-VLP Not applicable NCT03089788 Completed R. Mellid-Carballal et al. European Journal of Pharmaceutical Sciences 189 (2023) 106558 16 Bruckman et al., 2014). Another example are CCMV-VLPs, which can bind gadolinium (3+), resulting in paramagnetic nanoparticles useful for MRI (Allen et al., 2005). The two basic types of MRI images are T1-weighted and T2-weighted images. T1 shortening agents create a bright signal, while T2 agents originate a dark one (Koudelka et al., 2015). Iron oxide nanoparticles (T2 agents) have been displayed on bacteriophage M13 VLPs for in vivo imaging of prostate cancer (Ghosh et al., 2012). They have also been encapsulated into VLPs derived from the brome mosaic virus for imaging Nicotiana benthamiana plants (Huang et al., 2011). On the other hand, P22 capsids loaded with paramagnetic manganese (III) protoporphyrin IX complexes demonstrated potential as a T1-enhancing contrast agent (Qazi et al., 2014). MRI and NIR proved useful for real-time imaging of the biodistribution of innovative virosome-based systems. Precisely, EMHVs were used for drug delivery to the lungs, achieving effective targeting after intravenous injection thanks to an external magnetic field. Realtime imaging, together with magnetic-guided targeting, renders this system a fascinating example of a theragnostic tool (Vizzoca et al., 2022). New MRI contrast approaches, such as chemical exchange saturation transfer (CEST) and hyper-CEST imaging can also benefit from ViP NPs (Koudelka et al., 2015). The combination of icosahedral bacteriophage MS2 capsid and filamentous bacteriophage fd with xenon-based sensors are representative examples (Meldrum et al., 2010; Palaniappan et al., 2013). 4.1.3. ViP NPs in PET MS2-VLPs with DOTA groups covalently linked to their inner surface were used to chelate 64 Cu radioisotopes for PET. This system allowed imaging in mice for prolonged times, since the radioisotope remained for 24 hours in the bloodstream (Farkas et al., 2013). Pang et al. reported how PET can be used to monitor the delivery of drugs to brain tumors by CED. They fused green fluorescent protein to Qβ coat protein and produced fluorescently labeled VLPs in E. coli. These VLPs were loaded with epirubicin, modified with a cell-penetrating peptide, and labeled with 68 Ga-DOTA. CED infusion of the described system in animal models results in a synergistic therapeutic effect without systemic toxicity. PET/CT allowed to examine the distribution of multifunctional VLPs in the brain and their in vivo biodistribution in tumor-bearing mice (Pang et al., 2019). 5. Conclusions The discovery of VLPs and virosomes opened a wide spectrum of possibilities in the pharmaceutical field. ViP NPs take advantage of their viral nature but avoid safety problems related to the manipulation of viruses. Throughout this review, we made an overview of the progress made on ViP NPs. Up to now, prophylactic vaccination against viral infections benefited the most from ViP NPs. Additional ongoing research pursues innovative vaccination strategies which aim to simplify vaccine production and administration, thereby reducing costs and reaching developing countries. In addition, therapeutic vaccination with VLPs and virosomes showed promising results in clinical trials to treat cancer and other chronic diseases. Current research is also investigating the possibilities that ViP NPs offer for drug/gene delivery and for diagnostics, biosensing, and disease monitoring. The data suggest that ViP NPs have intriguing cell targeting and intracellular transport capabilities that merit further study. In summary, ViP NPs are already important tools in the pharmaceutical arsenal. It is envisaged that as new advances in biotechnology, synthetic biology, and nanotechnology are implemented in the following years, these fascinating platforms will extend to new therapeutic areas and reach new milestones in clinical use. CRediT authorship contribution statement Rocio Mellid-Carballal: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing – original draft. Sara Gutierrez-Gutierrez: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing – original draft. Carmen Rivas: Writing – review & editing, Supervision, Project administration. Marcos Garcia-Fuentes: Conceptualization, Writing – review & editing, Supervision, Project administration. Declaration of Competing Interest The authors declare no conflicts of interest. Data availability Data will be made available on request. Acknowledgments This publication is part of the following grants: PID2021–124986OB- I00 (Project “IMMMA”) funded by MCIN/AEI/ 10.13039/ 501100011033 and by “ERDF A way of making Europe”; AC21_2/00046 (Project “RAIN”) funded by Health Institute Carlos III/MCIN/AEI/ 10.13039/501100011033 and by “European Union NextGenerationEU/ Table 5 ViP NP-based systems under preclinical evaluation for in vivo imaging. Application Imaging technique Cargo External moieties Vehicle Targeted delivery of anticoagulant drugs to atherosclerotic plaques NIR NIR quantum dots Hirulog (drug) Targeting peptides for VCAM-1, macrophages, and fibrine SV40-VLPs In vivo imaging (vasculature, tumor resection…) NIR CH1/CH2 fluorophores – SV40-VLPs In vivo imaging of atherosclerotic plaques Dual optical and MR imaging Sulfo-Cy5-azide dye Gd-DOTA VCAM-1 targeting ligands PEG TMV nanoparticles Targeted in vivo imaging of prostate cancer MRI – SPARC binding peptide Iron oxide nanoparticles Bacteriophage M13-VLP In vivo biodistribution NIR fluorescence MRI – IRDye800CW EMHVs In vivo biodistribution PET-CT 64 Cu-DOTA PEG MS2-VLPs Glioblastoma treatment PET- fluorescence dualimaging Epirubicin 68 Ga-DOTA Cell-penetrating peptide Green fluorescent Qβ- VLPs R. Mellid-Carballal et al. 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