**2. Plant VLP-Derived Vaccines against Infectious Diseases**

The efforts of the vaccine industry have resulted in a comparably long list of licensed prophylactic vaccines that effectively prevent infection by different infectious agents (Supplementary Table S1). However, the efficacy of current vaccines is moderate or even poor in some cases and does not ensure long-lasting protection and the eradication of the corresponding diseases [39]. Additionally, there is an urgent need for vaccines against viral and bacterial pathogens, such as human immunodeficiency virus, tuberculosis, malaria, and many other agents, as well as emerging diseases, such as Zika virus, Dengue, Ebola and SARS. Therefore, interest in alternative solutions for the generation of new vaccines and the improvement of existing vaccines is continuously growing.

Experimental vaccines based on plant viruses have been constructed and tested for more than 30 years since the first TMV-based vaccine was developed against poliovirus [20]. In this chapter, we will discuss the recent development of plant virus-derived prophylactic vaccines against important pathogens such as influenza and malaria.

In most cases, plant virus-derived vaccines are peptide vaccines that are genetically fused or chemically coupled to viral CP. Peptide vaccines can stimulate the formation of neutralizing antibodies and ensure protection against the corresponding pathogen in animal models (Suppl. Table S2). The advantages of this approach include the reduced influence of short peptides on the structural integrity of viral carriers and the easy production of experimental vaccines. However, the antigen is frequently not a linear AA sequence but a spatially complex structure involving different parts of the antigenic protein. For such antigens, peptide-based vaccines are not suitable for stimulating protection via the immune response. Therefore, several authors have suggested the usage of whole proteins as antigens, which can result in epitope structures closely related to native ones and elicit neutralizing antibodies after immunizations. Genetic fusions of whole antigenic proteins with viral CPs negatively influence VLP formation in most cases. However, sometimes plant viral coats are able to accommodate even very long antigenic sequences on their surfaces [40,41]. As alternatives to genetic fusions, other approaches can result in antigen presentation, such as chemical coupling or physical binding of the antigen to the VLP surface using binding partner molecules. Successful examples of the generation of vaccines using the chemical or enzymatic coupling of whole antigens include studies of experimental vaccines against *Francisella tularensis* [42], influenza [43], *Yersinia pestis* [44], *Plasmodium vivax* [45], and Zika virus [46].

Methods used for the incorporation of whole antigen sequences into viral *CPs*, allowing the preservation of viral morphology, are discussed in recent review articles [25,47].

Several plant virus-based vaccines have been tested for tolerability, safety and efficacy in human clinical trials. The first such vaccine is an edible vaccine against rabies containing a recombinant antigen derived from rabies virus proteins (a fusion of peptides derived from the G and N proteins) incorporated in the N-terminus of the Alfalfa mosaic virus (AlMV). Introduction of the chimeric gene into the TMV-based plant vector and transient expression in spinach leaves resulted in AlMV-like particles exposing rabies epitopes. The inclusion of raw spinach leaves containing these VLPs in the diet of human volunteers led to significant antibody responses to rabies virus and plant virus carriers [48,49]. This pioneering study clearly demonstrates the potential of plant virus-based carriers for the generation of human vaccines. Later, several vaccine candidates were constructed based on AlMV carriers (Figure 3A [41,50]).

**Figure 3.** Examples of recombinant vaccine platforms used for introduction of different antigens in plant viral structures. (**A**) «launch» vector [51] based on TMV for production of antigen-containing viruses in plants. LB and RB denotes agrobacterial shuttle vector T-DNA region left and right borders; P35S—CaMV promoter; sgP—subgenomic promoter; T—transcription terminator; Rep, MP—TMV replicase and movement protein; pink and turkish arrows denote the relative position of antigen (A) and AlMV coat protein gene (*CP*), respectively [41]. (**B**) BaMV-based vector for production of antigen (A)-containing mosaic viruses in plants. *sgP*—subgenomic promoter; A—antigen-coding sequence; 2A—self-processing peptide sequence; *Rep*, *TGB*, *CP*–replicase, triple gene block and coat protein genes [40]. (**C**) Bacterial system for antigen introduction in plant VLP structure [28]. Antigen (A) and CMV VLPs are obtained from separate bacterial cultures, purified, derivatized and joined in chemical coupling reaction. CMV *CP* gene contains T-cell epitope coding sequence (red box).

Malaria is one of the biggest health threats, causing hundreds of millions of cases and 435,000 deaths in 87 countries, especially in Africa [52]. The existing vaccine, RTS,S, has demonstrated only limited efficacy in clinical trials [17]. A recent publication [53] summarizes the results of a Phase I study assessing a malaria transmission-blocking vaccine based on a recombinant fusion of the *P. falciparum* antigen Pfs25 with the plant virus AlMV. Vaccine candidates produced in whole plants under cGMP conditions demonstrate acceptable safety and tolerability and induce antibodies against Pfs25, depending on the injected vaccine dose. However, the generated antibodies did not produce the expected reduction in *P. falciparum* transmission from mosquitoes to human host cells. The authors concluded that it is necessary to improve the formulation of the vaccine and increase the proportion of the Pfs25 antigen incorporated in plant VLPs.

Recently, other plant virus-derived VLPs have been suggested as active components for vaccines against malaria. The authors recommend the usage of thrombospondin-related adhesive protein (TRAP) as an antigen, which is necessary for *Plasmodium vivax* sporozoite motility and liver cell invasion. The vaccine was designed based on Cucumber mosaic virus (CMV) VLPs with chemically coupled TRAP antigen that were formulated with microcrystalline tyrosine (MCT) as an adjuvant. Vaccine formulations containing VLPs increased antibody production against TRAP compared to that produced by the antigen alone. The VLP-based vaccine formulated with MCT conferred significant protection in the challenge test with recombinant *P. berghei*, suggesting that MCT can be used as an advantageous adjuvant alternative for prophylactic VLP vaccines [45].

The next most important viral infection is influenza, which causes 250,000 to 500,000 deaths globally every year. Today, there are 26 licensed inactivated vaccines, some of which are produced routinely. However, existing vaccines do not ensure complete protection against the circulating influenza strains [54]. The reasons for low vaccine efficacy are the low levels of hemagglutination-inhibiting antibodies and influenza strain variations in the hemagglutinin (HA) sequence. For the improvement of flu vaccines, the use of stronger adjuvants and increased amounts of HA as well as the addition of new antigens (neuraminidase and matrix protein M2e) to influenza vaccines should increase efficacy [39].

Different peptide antigens, including peptides derived from nucleoproteins and M2e proteins, have been tested using plant viruses as vaccine carriers and adjuvants. Some of them demonstrated significant protection against challenges in different animal models (see examples in Supplementary Table S2). One experimental vaccine is currently being tested in a human trial that aims to evaluate the safety and reactogenicity of Papaya mosaic virus (PapMV) VLPs as adjuvants for use in a seasonal flu trivalent vaccine in 48 healthy volunteers [55]. The efficacy of the plant VLP-adjuvanted flu vaccine has not yet been reported. Earlier publications suggest that filamentous PapMV VLPs containing a consensus peptide of the influenza matrix M2e protein increase the survival of immunized mice if used as an adjuvant together with a trivalent inactivated flu vaccine. These results demonstrate the strong adjuvanting properties of filamentous plant VLPs for influenza vaccines [56].

Plant virus-based peptide vaccines are also being evaluated in veterinary trials. A vaccine based on Cowpea mosaic virus (CPMV) protects vaccinated dogs against lethal challenge with canine parvovirus [57]. Another vaccine against foot and mouth disease virus (FMDV) was designed using a bamboo mosaic virus (BaMV) vector and 37 AAs from the FMDV VP1 protein; all immunized swine were protected against FMDV challenge [58]. Another veterinary trial demonstrated vaccine safety and the protection of pigs against porcine circovirus (PCV) after vaccination with an inactivated CMV-based vaccine containing an incorporated PCV CP epitope [59].
