**3. Plant VLP-Derived Anticancer Vaccines**

Cancer is one of the leading causes of death globally and was responsible for an estimated 9.6 million deaths in 2018 [60]. Currently, alongside surgery, chemotherapy and radiation, cancer immunotherapy has become an important component of cancer treatment.

Cancer immunotherapy has a more than 150-year history. It began with the first observations of the significant regression of sarcomas in patients after accidental infection by a *Streptococcus* bacterium. In 1891, William Coley, an American surgeon, used heat-inactivated bacteria to treat a large number of patients suffering from inoperable cancers. His therapy method resulted in the cure of more than 1000 patients. However, the method was later replaced with radiation and chemotherapy due to dangerous infection risks and the absence of reproducible results. The next important milestone in the development of cancer immunotherapy was the finding of Old et al. in 1959, which demonstrated the antitumor activity of the tuberculosis vaccine (BCG) in a mouse model. The vaccine has been introduced for the treatment of bladder cancer and has been used in clinics since the 1970s. These and other key events in cancer immunology are discussed in several recent review articles [61,62].

Several cell- and protein-based anticancer vaccines are approved for use in clinics or are being tested in late-stage clinical trials, such as autologous dendritic cell vaccines, recombinant virus-based vaccines, peptide-based vaccines, DNA vaccines, and human tumor whole-cell vaccines. However, for the most part, clinically tested anticancer vaccines have demonstrated limited or no efficacy in comparison to that of traditional treatments, requiring the development of new strategies and combinations of different approaches [63,64].

One such new approach for vaccination against cancer is based on the usage of nanoparticles, such as liposomes, carbon nanotubes, synthetic biodegradable and biocompatible polymers, inorganic nanoparticles, VLPs and different combinations of such particles [65].

VLPs represent a powerful and flexible tool for generation of the active components of cancer vaccines, as demonstrated in numerous preclinical studies (for review see [66]). We have already discussed the advantages of VLPs as vaccines, such as the multiplicity of antigens, the sizes of VLPs, which allow them to enter the lymphatic system, and their capability to encapsulate nucleic acids stimulating Toll-like receptors. As a result, VLPs are able to induce strong T cell responses, which is the most important requirement for a therapeutic vaccine against cancer. Moreover, VLP technology is already used for the prevention of cancer with Papilloma virus-derived VLPs, which efficiently protect immunized individuals against cervical cancer [66].

Plant viruses, including both native and recombinant viruses as well as their noninfectious derivatives (VLPs), have been considered as nanoparticle structures with antitumor activity since 2006, when McCormick et al. introduced a melanoma-specific peptide into TMV using recombinant fusion and chemical coupling [67,68] and tested both vaccines in tumor challenge models. The authors observed direct TMV uptake by dendritic cells and enhanced production of interferon È (IFNg). Interestingly, the vaccine prepared by the chemical coupling of peptides to TMV ensured better survival of the animals than the recombinant vaccine when both vaccine variants were formulated with the CpG DNA adjuvant.

The importance of vaccine formulation was also demonstrated in a recent study using icosahedral CMV VLPs with chemically coupled p33 peptide derived from lymphocytic choriomeningitis virus (LCMV). The vaccine was formulated with MCT, CpG and alum adjuvants and tested in an aggressive mouse melanoma model.

VLPs adjuvanted with MCT effectively retarded the development of the tumor. The effect was comparable when CpG DNA was used as the adjuvant, whereas alum was ineffective in slowing down tumor growth [69].

A recent study suggests a highly interesting immunotherapy approach. Three different icosahedral plant virus-derived nanoparticles with chemically coupled breast cancer epitopes efficiently elicited the formation of antibodies against the HER2 receptor, which is overexpressed in breast cancer cells. Additionally, all three vaccines stimulated T-cell-mediated immune responses when tested separately. Sequential use of these vaccines reduced the immune responses against VLP carriers and improved the formation of antibodies against the HER2 peptide. This suggested prime-boost strategy considerably reduced tumor development and enhanced the survival rate in a mouse tumor model. The study clearly demonstrated that the Th1-type immune response, including the formation of IgG2a antibodies, the secretion of IFNg and the activation of CD4+/CD8+ T-cells, is the most important factor for immunotherapy. Interestingly, plant VLPs derived from CPMV demonstrated better immunotherapeutic properties than those derived from Cowpea chlorotic mottle virus (CCMV) and Sesbania mosaic virus (SeMV). Therefore, additional studies are needed to understand the differences between different plant VLPs used as carriers for anticancer vaccines [70]. Other examples of plant virus-based cancer vaccines with the corresponding literature citations are summarized in Supplementary Table S3.

The analysis of several recent publications reveals the potential application of unmodified plant VLPs without any introduced antigens as cancer immunotherapy agents [71,72]. In one study, the authors compared differently prepared nanoparticles derived from CPMV as immunotherapeutic agents in a murine ovarian cancer model. Native CPMV particles containing viral RNA induced a more pronounced therapeutic effect and the survival of experimental animals in comparison to empty CPMV VLPs produced in plant or insect cells. The enhanced immunomodulatory effect apparently is due to the presence of encapsidated ssRNA in native CPMV virions, which activates Toll-like receptors 7/8. Interestingly, the chosen recombinant host can also influence the immune-stimulating properties of produced plant VLPs [72].

In another study, structural variants of plant virus-derived nanoparticles from several species were used as in situ vaccines, such as icosahedral CPMV and three variants of TMV (native, in vitro RNA-templated assembly of short TMV, and spherical TMV produced after thermal treatment of native virions). The results suggest the superior immune-stimulating properties of CPMV compared with those of other structural variants of TMV and confirm that antigen multiplicity is one of the most important factors involved in eliciting a strong immune response. One possible explanation for the enhanced immune-stimulating properties of CPMV in a mouse melanoma model is the ability of VLPs to recruit monocytes into the tumor microenvironment, leading to the infiltration of neutrophils and natural killer cells and resulting in tumor growth inhibition. Authors suggest that the intrinsic properties of some plant viruses allow them to be developed as cancer vaccines for clinical use; however, the detailed mechanisms of immune stimulation remain to be elucidated [71].

#### **4. Plant VLP-Derived Vaccines against Allergies, Autoimmune Diseases and Other Diseases**

Vaccinations are also highly promising for the treatment of allergies and autoimmune and neurodegenerative diseases, as demonstrated in the latest studies. Similar to antimicrobial and anticancer vaccines, plant viruses and VLPs can serve as carrier structures for corresponding antigens, which are important in disease development.

One of the most challenging diseases in terms of therapy development is Alzheimer's disease (AD). More than 40 million people worldwide are suffering from dementia, which is caused by the formation of plaques containing proteolytic fragments of amyloid precursor protein Aβ (AA 1-42). Existing therapies and experimental approaches, including the use of monoclonal antibodies and vaccines, have only a small impact on disease progression. According to a recent opinion, vaccination should be prophylactic; the levels of specific antibodies have to be sufficiently high for the targeting of oligomeric species of Aβ peptides, and the sizes of the epitopes included in vaccines have to be less than 8 AA to prevent the stimulation of pathogenic T-cell responses [73].

The concept of AD vaccines has existed for more than 15 years; however, the first clinical trial using a vaccine containing aggregated Aβ peptide (AA 1-42) resulted in cases of T-cell-mediated brain inflammation [74]. Later, an experimental vaccine consisting of Aβ peptide and a bacteriophage Qβ conjugate produced strong antibody responses without significant T-cell responses in mice [75].

Plant viruses and derived VLPs are also considered as epitope carriers for Alzheimer vaccines. Potential vaccine candidates have been generated using infectious CMV fused with Aβ peptides purified from plant biomass [76]. Recently, we constructed a plant VLP platform (Figure 3C) based on the same CMV by the genetic incorporation of a universal T-cell epitope in the interior of particles and the chemical coupling of Aβ1-6 peptide to the VLP surface. Sera obtained from immunized mice were shown to recognize Alzheimer plaques in human brain sections, suggesting that the CMV-Aβ(1-6) vaccine induced the production of specific antibodies [28].

Allergic disorders have been among of the most common chronic diseases in Europe in recent decades [77]. Patients suffering from allergies have to avoid allergens and use anti-histamine medications. Alternatively, allergen-specific immunotherapy (AIT) is the only available treatment for the reduction of allergy symptoms. For therapy, crude allergen extracts containing a mixture of native allergenic proteins are used. The typical disadvantages of AIT are the risk of anaphylactic reactions, the long duration of therapy, the low quality of the natural extracts used in AIT and the unsatisfactory efficacy of the treatment.

Peanut allergy is the most frequent cause of anaphylactic reactions and death among food allergies. There is currently no safe and effective therapy for peanut allergy, especially for patients with a severe allergy. Recently, we constructed several plant virus-based, immunologically optimized vaccines for peanut allergy by chemical coupling of peanut allergens Ara h 1 and Ara h 2 as well as mixture of proteins purified from roasted peanut extract to CMV VLPs. The resulting vaccines did not cause allergic reactions and induced specific IgG antibodies to protect peanut-sensitized mice against anaphylactic shock. Notably, immunizations with single allergen-containing VLPs ensured protection against challenge with the complex allergen mixture, suggesting a new vaccination strategy for the treatment of peanut allergy [78]. Other examples of VLP usage in the treatment of allergic diseases are summarized in a recently published review article [79].

Allergies to cats affect more than 10% of the human population, and the prevalence is increasing [80]. Similar to that of food allergies, current cat allergy treatment includes the avoidance of exposure, the use of anti-histamines and steroids and long-term subcutaneous immunotherapy. All these measures are only partially effective in eliminating allergic reactions; additionally, immunotherapy is bound to result in safety issues. The predominant cat allergen is the secretoglobulin Fel d 1, which is secreted by cat sebaceous and salivary glands. We generated a CMV-based vaccine containing recombinant Fel d 1 allergen using chemical coupling and demonstrated that VLP coupling effectively reduces allergic reactions, stimulates the formation of Fel d 1-specific IgGs and protects sensitized mice against anaphylactic shock [76]. Moreover, Bachmann and collaborators proposed a new strategy for the

treatment of Fel d 1 allergy involving immunizing cats against their own Fel d 1 allergen. The induced anti–Fel d 1 antibodies exhibited a strong neutralization ability and might result in reduced symptoms in allergic cat owners [81].

Monoclonal antibodies (mAbs) produced by immortalized hybridoma cells represent a new way to treat different diseases. Since the approval of the first mAb preventing kidney transplant rejection in 1992, numerous mAbs have been used in clinics for treatment of cancers, bacterial and viral infections, and various cardiovascular, respiratory, neurological and autoimmune diseases [82,83]. MAbs have been proven to be efficient agents for treating chronic inflammatory diseases via the selective inhibition of cytokines, which are excessively produced in several disease conditions. Taking into account the high costs and side effects of mAb therapies, there is significant interest in replacing mAb treatments with active immunization against autologous proteins, including interleukins [84,85].

Based on this idea, we generated an IL17-containing plant VLP-based vaccine and tested it in a mice psoriasis model [28]. The vaccinated mice demonstrated a similar reduction in psoriatic symptoms compared to that in animals passively immunized with IL17A antibody. Moreover, the vaccination elicited protective effects in suboptimal conditions, such as those involving older mice or low vaccine doses.

The knowledge obtained from successful studies of human vaccinations and mAb treatments can be transferred to the development of veterinary vaccines. It is well known that, for example, allergic hypersensitivity is linked to the activation of eosinophils and the enhanced production of interleukin 5 (IL5) by Th2 cells. The eosinophil count can be effectively reduced by a specific humanized anti-IL5 mAb, which is used for treatment of human asthma [86]. Active vaccination with a plant VLP-IL5 conjugate instead of a mAb induced a potent IL5 antibody response in horses, reducing the symptoms of insect bite hypersensitivity. Simultaneously, vaccination did not significantly influence the blood eosinophil count and the parasitic load in vaccinated horses [87,88].

Another example of a CMV-based therapeutic vaccine is a vaccine reducing the production of IL-31 in dogs and horses suffering from itching during atopic dermatitis or insect bite hypersensitivity. In both animals, the vaccine is well tolerated and improves the disease symptoms [89,90].

In the last few years, nerve growth factor (NGF), a key molecule involved in the regulation of neuronal regeneration during injury and pain perception, has been suggested as a promising target for osteoarthritis (OA) treatment. In humans, monoclonal antibodies against NGF significantly suppress pain associated with late-stage OA. Based on this, we constructed a CMV-based NGF vaccine and demonstrated its therapeutic efficacy by showing that it alleviates spontaneous pain behavior in surgically induced murine OA [91].
