**1. Introduction**

Vaccination is one of the most powerful public health interventions of the 20th century, preventing an estimated six million deaths a year [1]. From a public point of view, vaccination programs result in cost savings that exceed investments by 16-fold [2].

During the last 200 years, academic scientists and the vaccine industry have developed a large number of vaccines, including approximately 50 vaccines licensed for human use and more than 300 veterinary vaccines [3]. All currently used vaccines can be classified into several types, and the classification also reflects the historical steps in vaccine development [4,5]. These types include vaccines against different infectious agents (bacteria, viruses, parasites); vaccines obtained by different methods of development; vaccines containing single or multiple antigens and recombinant vaccines (Figure 1, Supplementary Table S1).

**Figure 1.** Classification of vaccines. (**A**) vaccines against different infectious agents; (**B**,**C**) vaccine types according to method of development; (**D**) vaccines containing single or multiple antigen subtypes from the same pathogen (mono-or poly-valent); (**E**) vaccines containing native or artificially generated (recombinant) antigens. The proportion (%) of a corresponding vaccine type is calculated based on the list of diseases and available vaccines (Supplementary Table S1; based on data from World Health Organization [6]).

The very first vaccine was the smallpox vaccine introduced by Jenner more than 200 years ago [7]. He used a heterologous virus containing material originating from infected cows (cowpox) for the protection of humans against smallpox. Further development and international use of the vaccine led to the complete eradication of smallpox, which is one of the greatest achievements in medicine [4].

Targeted vaccine development began with Luis Pasteur's discovery that pathogenic microorganisms are less virulent if cultivated under suboptimal conditions or treated with different chemicals. He introduced the concept of attenuated vaccines, which was later exploited in the generation of many vaccines, including vaccines against anthrax and rabies [4]. Later, progress in cell culture technology enabled the culture of mammalian cells and allowed the propagation of viruses, resulting in effective attenuated vaccines against polio, measles, mumps, rubella, influenza and other infectious diseases [8].

The third group of vaccines was generated by the chemical inactivation of infectious bacteria and viruses as well as bacterial toxins (toxoid vaccines). The application of increased temperatures or inactivating chemicals in the production of different vaccines is still used by industry for vaccines against cholera, polio, rabies, Japanese encephalitis and other infectious diseases [9].

The development of molecular genetics, bacteriology and biochemistry contributed significantly to the understanding of the molecular structure of living cells and allowed the identification of pathogen components potentially suitable for vaccine generation. The exploitation of this idea resulted in a long list of so-called subunit vaccines, including vaccines against *Haemophilus influenza,* meningococcus, pneumococcus (polysaccharides and their conjugates with carrier proteins) and hepatitis B (plasma-derived protein antigen) [9].

The advent of molecular biology resulted in the next breakthrough in the medicinal industry. The achievements in gene engineering have dramatically influenced the construction of vaccines. Since the 1980s, recombinant technologies have been introduced for the development of different vaccine types, such as live and attenuated recombinant bacteria and viruses, as well as the production of toxins and other protein antigens using recombinant hosts [8].

The hepatitis B (HBV) vaccine was the first subunit vaccine, and it was generated by gene engineering techniques more than 30 years ago. The expression of a cloned copy of the HBV surface antigen (HBsAg) in yeast cells resulted in the production of noninfectious virus-like particles (VLPs) [10] and allowed the replacement of a previously generated plasma-derived vaccine [11]. The success of HBV vaccines strongly stimulated the development of recombinant vaccines based on viral structural proteins and marked the beginning of a new era in rationally designed VLP platforms for the generation of prophylactic and therapeutic vaccines [12].

Using the same principle of viral coat protein (CP) expression in heterologous hosts, vaccines against cervical cancer (Gardasil and Cervarix; [13–15]), hepatitis A (Hecolin; [16]) and malaria (RTS,S; [17]) were constructed, clinically tested and licensed for human use in the subsequent decades. It is important to stress that the RTS,S vaccine is the first among licensed vaccines containing a VLP carrier (HBsAg) with an incorporated foreign antigen (CS).

Artificial virus-like structures derived from plant virus proteins are well known due to virus assembly studies performed since the 1950s [18]. Based on the use of carrier proteins with chemically coupled peptide antigens as promising vaccine candidates [19], Haynes et al. [20] generated an experimental vaccine using a gene engineering approach instead of chemical coupling. They combined the tobacco mosaic virus (TMV) *CP* gene with an extension encoding a C-terminally located, 8 AA-long antigenic peptide from poliovirus. The resulting VLPs purified from recombinant bacterial cells were immunogenic and stimulated the formation of antibodies against poliovirus in rats. These results, together with those of HBsAg [21], introduced the use of VLPs as a central carrier element of many experimental and licensed vaccines. Additionally, the study clearly demonstrates that nonpathogenic viruses are also suitable for vaccine generation after the introduction of relevant antigens into their structure.

Viruses and their derivatives possess several characteristics that are highly important for their use as vaccines [22]. Most likely, the most important property of viruses and VLPs is their structural organization. Structurally, viruses are constructed of hundreds or thousands of highly ordered CP molecules, which serve as repeated antigens for the mammalian immune system. These antigens on the virus surface can stimulate B cells by crosslinking B cell receptors and induce long-lasting antibody responses. In addition, most viruses have the optimal size, shape and rigidity to enter the lymphatic system through the pores in lymph vessels. This facilitates the trafficking of viral particles and VLPs to lymph nodes and their uptake by antigen-presenting cells (APCs). Moreover, viruses and VLPs encapsulate specific host-derived nucleic acids (DNA or RNA), which stimulate Toll-like receptors in APCs and serve as natural vaccine adjuvants [23,24].

Plant viruses and VLPs (Figure 2), compared with other VLPs, have additional advantages as vaccine carrier structures. It is well known that plant viruses are not able to infect mammalian organisms. Therefore, the probability of pre-existing immunity against plant viruses is considerably lower compared with that against VLPs derived from mammalian viruses (e.g., HBV and papilloma virus). Most plant

viruses are assembled from single or a few CP molecules and demonstrate remarkable structural flexibility, allowing different manipulations, such as disassembly/reassembly, as well as chemical and genetic modifications. This enables the rational design of vaccines and the introduction of different antigens derived from infectious agents, allergens and self-molecules. Additionally, plant VLPs and other artificially generated VLPs do not contain replicating nucleic acids. This prevents the reversion of vaccines into infectious viruses, which is a serious risk factor for attenuated viral vaccines. From a technological viewpoint, plant viruses and VLPs can be produced in different recombinant hosts (bacteria, yeasts, plants, and eukaryotic cells) under cGMP conditions; the mentioned structural flexibility of plant viruses allows the construction of universal vaccine platforms [25,26].

**Figure 2.** Examples of icosahedral and helical plant viruses, used for vaccine development. Images were created using Protein Data Bank and NGL 3D viewer [27]. α-helices are shown in red, β-sheets–in yellow. (**A**) Cucumber mosaic virus (CMV) structure (T = 3 symmetry, diameter = 28 nm). Image of 5OW6 [28]. (**B**) Cowpea chlorotic mottle virus (CCMV) structure (T = 3 symmetry, diameter = 29 nm). Image of 1CWP [29]. (**C**) Cowpea mosaic virus (CPMV) structure (T = 3 symmetry, diameter = 28 nm). Image of 1NY7 [30]. (**D**) Sesbania mosaic virus (SeMV) structure (T = 3 symmetry, diameter = 28 nm). Image of 1X33 [31]. (**E**) Tobacco mosaic virus (TMV) structure (cryo-EM reconstruction of a TMV fragment; particle length = 300 nm, diameter = 18 nm). Image of 3J06 [32]. (**F**) Bamboo mosaic virus (BaMV) structure (cryo-EM reconstruction of a BaMV fragment; particle length = 490 nm, diameter = 15 nm). Image of 5A2T [33].

In this review, we summarize the publicly available data on vaccines derived from plant viruses, emphasizing the newest developments in the construction of experimental vaccines. We used two databases as a source of information: the PubMed database of the US National Institutes of Health (https://www.ncbi.nlm.nih.gov/pubmed/) and the Web of Science (https://apps.webofknowledge.com). Our literature search demonstrates the rapidly growing interest in plant viruses as vaccine carriers; for example, a cumulative search for the term "plant virus vaccine" in Web of Science yielded 787 publication records through 2009, more than 1400 through 2014, and at least 2204 publications through the end of 2019. As revealed by the search, plant virus derivatives serve as components for at least 71 experimental vaccines against infectious diseases, 16 anti-cancer vaccines and 10 vaccines against allergies and autoimmune diseases (examples and literature citations are summarized in

Supplemental Tables S2, S3 and S4). The increasing interest and recent publications stimulated us to summarize and discuss the latest developments in the design of vaccines based on plant viruses. Descriptions of other plant VLP applications can be found in several recent review articles [34–38].
