Traditional vaccines, such as inactivated or live-attenuated, have shown protective efficacy against several infectious diseases that are caused due to viral infections. However, they sometimes have safety concerns and deviations in the protective immune response. Recent advances in nucleic acid-based, subunit, and recombinant viral vector-based vaccines may overcome the limitations of traditional vaccines against infectious pathogens by enhancing immunity without compromising safety, efficacy, and tolerability and can also control infectious diseases during outbreaks or pandemics. The significance of such vaccine approaches in rapidly developing vaccine candidates for pandemic threats has been previously reported and is considered an approach for pandemic preparedness and response (1). Virus-like particles (VLP) differ from other subunit vaccines in that they have strong immunogenicity because they present repetitive antigenic epitopes to the immune system in more authentic confirmation and move in parallel with viral vector-based vaccines to produce protective, effective, and efficient vaccines against severe infectious diseases (2).

VLPs are non-infectious multiprotein structures that mimic native viruses but lack the disease-causing viral genome, making them safer vaccine candidates. Because of their optimal size (20–200 nm), they can practically display any epitope in a multivalent format that guarantees a high humoral or cell-mediated immune response in the host.

VLPs are categorized into enveloped and non-enveloped based on the presence or absence of lipid envelopes. Furthermore, these particles can be classified as homologous or heterologous according to their composition. Homologous VLPs contain proteins derived only from the native viruses. On the other hand, heterologous VLPs contain proteins from different sources, which increases their immunogenicity (3). They are further classified as single- or multi-capsid proteins. Simple VLPs are composed of a single capsid protein that is expressed in both eukaryotic and prokaryotic systems. In contrast, multi-capsid proteins show several striking structural characteristics, including many complicated concentric layers of different capsid proteins. Multi-capsid VLPs are usually produced in eukaryotic expression systems such as yeasts, insect cells, and plants. For example, various combinations of four different rotaviral capsid proteins can produce stable VLPs with double or triple capsid layers (4). Complex multi-capsid VLPs present higher production challenges. In this case, each protein should be expressed in stoichiometric amounts for efficient production because of limited cellular resources, as the expression of excess monomers does not improve VLP assembly (5). Enveloped VLPs (eVLPs) are poorly characterized at the biophysical level because they are structurally less uniform than non-enveloped VLPs. Technical challenges remain in the generation of eVLP-based vaccines in their design, treatment, and storage. Proper folding and glycosylation of viral surface antigens are important for defining the efficacy of eVLP-based vaccines because they are critical for stability, immune recognition, and pathogenicity. In general, eVLPs with host-derived envelopes are more sensitive to the external environment than non-enveloped VLP. Changes in conditions, such as temperature, shear force, and purification processes, can destroy the integrity and stability of particles, reducing eVLP immunogenicity. However, computational design, in vitro assembly of VLPs, removal or replacement of the transmembrane region, introduction of a suitable heterologous signal peptide, new bioprocessing modalities, and new macro-scale purification materials can address these problems robustly, at the expense of process complexity (6).

VLPs carry pathogen associated molecular patterns (PAMP), which are sensed by pathogen recognition receptors (PRRs) present either on the cell surface or endosomes of dendritic cells (DC). Presentation of VLP-peptides along with major histocompatibility complex (MHC) class I or class II molecules by DC induces CD8+ or CD4+ T cell responses, respectively (7). Simultaneously, co-stimulatory molecules present on DC activate T-helper (Th) cells, which are required for both cell-mediated and humoral immunity. In some cases, B cells are activated without innate immunity or any direction by Th cells (6). The addition of more immunogenic domains in VLP could further enhance their potency and efficacy, as seen in the case of hepatitis B core antigen (HBcAg). HBcAg-based VLP vaccines contain more immunogenic domains of hepatitis B surface antigen (HBsAg) and stimulate broad and specific humoral and cell-mediated responses against hepatitis B virus (HBV) (8). Because VLPs elicit a protective response at lower doses, they can significantly reduce vaccine costs (9).

Several platforms have been used for VLP production. The choice of the preferred platform provides flexibility in the manufacturing conditions required for the scaled-up production of VLP vaccines (7). Remarkably, VLPs can be recombinantly produced by expressing either a single type of coat protein, multiple structural proteins, or a combination of structural and nonstructural proteins required for VLP production. Examples include the first commercial VLP-based vaccine licensed for the HBV (9). To date, hundreds of VLP-based vaccine candidates have been studied for immunization against various human and animal diseases. However, only a few have been commercialized to target non-enveloped viruses. The structure of non-enveloped VLPs consists of either a single or multiple structural protein(s) of a specific virus and is relatively simple. In contrast, eVLPs have host cell components that pose potential challenges to their regulatory approval, scalability, and stability (10). Both types of VLPs can be engineered to form chimeric VLP, which display foreign antigens to enhance immune response and can also be used as nanocarriers for the delivery of desired antigens or therapeutic component of a molecule (11).

This article briefly presents the merits and demerits associated with various platforms, recent advances, and technical challenges in VLP production. The current scenario of VLP-based vaccine candidates at commercial, clinical, and preclinical levels are also summarized.

VLP-based vaccines have been developed as the next-generation vaccines. However, to ensure optimal protein folding, post-translational modifications (PTMs), cost-effectiveness, and scale-up production of these vaccines in a timely manner, an appropriate platform must be selected. VLPs can be experimentally generated in the laboratory using recombinant viral proteins that are expressed in a range of expression systems including prokaryotic cells, yeast, insect cell lines, plants and mammalian cell lines. In addition, cell-free expression techniques have been used successfully. In these cases, VLP proteins are expressed first in a cell-based expression system, then assembled in a cell-free environment for proper folding (6). All systems have their own advantages and drawbacks, which are briefly listed in Table 1 .

The optimal expression of each VLP is usually identified via trial-and-error by comparing the translated products of multiple expression systems. Expression in E. coli is often preferred for producing small proteins with limited PTMs; however, PTMs of larger proteins require a more complex expression system, such as yeast, baculovirus, and mammalian cells. Compared to higher eukaryotes, yeasts are considered a cost-effective production system. In addition, yeasts avoid endotoxin and viral contamination problems associated with bacterial and mammalian expression systems (13). Engerix-B (HBV vaccine) and Gardasil (Human Papilloma Virus (HPV) vaccine) are two FDA-approved vaccines that have been generated in yeast expression systems. Despite these achievements, the lack of complex PTMs is a major drawback of yeasts, which limits their use in generating non-enveloped VLPs. In contrast, animal cell expression platforms are attractive because of their ability to make complex and precise PTMs that are essential for proper protein folding, which can be used to produce multiple structural proteins of non-enveloped and enveloped VLPs (6).

Due to the ease and speed of baculovirus-based VLP expression, this system is suitable for manufacturing vaccines against viruses that rapidly change their surface antigens between outbreaks, such as influenza virus (6, 14). Cervarix is an FDA-approved HPV vaccine produced using this expression system. The baculovirus/insect cell platform has also been used as a prophylactic vaccine candidate against several infectious diseases such as human immunodeficiency virus (HIV) 1, influenza virus A, chikungunya virus, severe acute respiratory syndrome (SARS), Ebola virus, dengue fever virus, Rift Valley fever virus (RVFV), Norwalk virus, and hepatitis C virus (HCV) (6, 15). The major drawback of the baculovirus/insect cell platform is its simpler N-glycosylation pattern compared to that of mammalian cells, which can be a disadvantage for some VLP applications.

VLP production in transgenic plants has several interesting applications. For example, although plant cells lack mammalian-like PTMs, plant-specific glycosylation can have an immunostimulatory effect (13). Plants have been used for VLP production of the Norwalk virus (16, 17), HIV-1 (18), and influenza virus VLPs (Medicago). Another recently developed expression platform is the cell-free system, which usually consists of extracts from E. coli or yeast cells. This system is preferred for the production of viral capsid proteins with toxic intermediate forms. Owing to the non-replenishing nature of a cell-free system, this method is highly demanding, with scalability limitations. Examples of commercialized VLP vaccines produced in a cell-free expression system include the influenza vaccine Inflexal V (19) and the hepatitis A vaccine Epaxal (20).

Three culture modes were used to produce the VLPs: batch, fed-batch, and continuous cultivation. Process design is of crucial importance because it influences yield, productivity, and final product concentration. The cost of raw materials can be decreased by maintaining a yield close to the maximum theoretical value. Simultaneously, an increase in high product concentration and productivity, and a reduction in non-productive periods can reduce the cost of downstream processing and contribute to the efficient use of manufacturing equipment. Owing to its simplicity and low risk of contamination, the batch mode is preferred for many industrial processes. In batch culture, all media components are added to the bioreactor at the start of the cultivation process, with no extra nutrients supplied until the end. However, the maximum potential of this process is often not achieved. Substrate and product inhibition, low virus titers, temperature sensitivity, low productivity (21) and ineffective use of time between batches are major problems in the batch mode. For efficient performance of batch fermentation, all non-productive steps (such as cleaning and filling of bioreactor, media formulation, etc.) should be minimized while targeting the maximum yield of the end product (22). This approach has been used to test a variety of VLPs, including HIV (23), chikungunya virus (24) and Ebola (25). In a fed-batch process, substrate inhibition is avoided by reducing the lag phase, while correctly designed feeding of concentrated nutrients increases product concentration and yield. The rate of the reaction is determined by the amount of limiting nutrients in the feed medium. In a baculovirus/insect cell expression system, this culture mode was tested for the production of parvovirus-like particles (26) and recombinant HBsAg (27). The highest productivity is achieved in continuous mode because the culture is in the log phase. In this mode, fresh medium is supplied and the conditioned medium is extracted. Continuous production is obtained using this method, and the product must be stored under proper conditions while being produced. This approach has been used for producing rabies VLPs in HEK293 cells (28). There are many types of continuous modes such as chemostat, turbidostat, stressostat, and morbidostat. Chemostat is the most widely used continuous cultivation method (29). It is possible to control the specific growth rates and product formation in a chemostat by changing the concentration of the limiting substrate for a specific strain. However, the increased risk of contamination, high volumes of low-concentration broth from the system, and low product concentrations are problematic (30). The fermentation process should be selected by considering the advantages and disadvantages of each process. The major criterion for the selection of a bioreactor/fermentation process is the minimal capital cost per unit product yield. For example, the design of a continuous production process for measles virus is more challenging because of its size (300 nm – 1 μm) and lysis of host cells. Cell lysis leads to rapid accumulation of host cell proteins (HCPs) and cell debris in the surrounding medium. Therefore, filtration during cultivation is challenging because the filter pores are blocked by debris or HCPs. Discontinuous filtration or repeated batch mode is more appropriate for this virus (21, 31). The corresponding yields from the different culture modes are summarized in Table 2 .

The bioreactors used in the manufacturing of VLP-based vaccines are made of stainless-steel vessels. However, single-use technology is gaining importance since it eliminates the need for in-situ cleaning and sterilizing, which reduces cross-contamination, investment, and operational costs. However, single-use vessels are less useful (14). Each of the three culture modes have several advantages and disadvantages ( Table 3 ).

Bacterial systems (mainly E. coli) are used to produce approximately 28% of all VLPs (43). In these systems, viral genes are codon-optimized for bacteria and cloned into commercial plasmids with strong promoters to generate higher yields and easy initial purification (44). However, a major drawback of these systems is their inability to induce post-translational glycosylation in the eukaryotic proteins and the endotoxins created during production of recombinant proteins. Little progress has been made in introducing glycosylation into E. coli-based cell-free systems. Asparagine-linked glycosylation is the most general and universal PTMs that determines the folding, stability, and catalytic activity of a recombinant protein. Protein glycosylation relies on the activity of pglB oligosaccharyltransferase (Pilin glycosylation B OTase), which transfers oligosaccharides to N-linked asparagine, thereby facilitating glycosylation (45, 46). In Campylobacter jejuni, a single pglB gene encodes all enzymes required for the biosynthesis of N-linked glycans. The promiscuity of C. jejuni PglB OTase has exhibited efficiency in E. coli (47). Subsequently, a glycoengineered E. coli strain, CLM24, was developed to express both Cj PglB OTase and Cj lipid-linked oligosaccharides (48, 49). The glycosylation components of the cell-free glycoprotein synthesis (CFGpS) system do not require a purification step and thus are easy to use and cost-efficient (48). The CFGpS system is the first prokaryotic cell-free system capable of cell-free transcription, translation, and glycosylation of proteins. Recently, a glycolic platform was developed that facilitates the glycosylation of VLPs in the E. coli cytoplasm (50). This glycolic platform uses the asparagine glucosyltransferase of Actinobacillus pleuropneumoniae, which is expressed in E. coli and catalyzes the transfer of single beta-linked glucose onto recombinant proteins. Similarly, to overcome the drawbacks of endotoxins, several genes such as LPS that participate in the expression of endotoxins are interrupted to eliminate endotoxin contamination and produce better recombinant proteins in E. coli, as shown in the case of the production of a 9-valent HPV vaccine candidate in E. coli ER2566 strain using a chromosomal integration approach, with markedly reduced residual endotoxin levels (51).

Insect, yeast, and mammalian platforms have also been used for the production of VLPs since the 1980s (43). Recombinant insect cells are a promising alternative platform to the baculovirus–insect cell system because target proteins can be produced simply by culturing the cells (52). For example, the secretory form of influenza VLPs consisting of hemagglutinin (HA) and matrix proteins was successfully produced by recombinant insect cells (53). Manipulating genes in yeasts is comparatively straightforward, and transformed cells can easily grow to extremely high densities. Therefore, VLPs can be produced in large volumes using commercial-scale fermenters. Among the many yeast species, Saccharomyces cerevisiae and Pichia pastoris have emerged as the most promising candidates for developing VLP-based vaccines. Noticeable changes in the production of VLP-based vaccines involve recombinant strategies that induce the secretion and production of multilayered VLPs, which are typically composed of more than one type of structural protein. For example, co-expression of four structural proteins in yeast expression systems effectively produces enterovirus 71 (EV71) VLPs (54). EV71 causes hand, foot, and mouth disease (HFMD), which is associated with severe neurological complications. VLP-based vaccines have shown great potential for preventing EV71 infections. Moreover, there have been a number of attempts to produce VLPs using new expression hosts, such as Hansenula polymorpha (methylotrophic yeast) is particularly striking (55). H. polymorpha has several unique characteristics such as thermostability, avoidance of hyperglycosylation, and use of several carbon sources (56). This platform has been established for the cost-effective production of VLPs at a larger scale (40 mg/100 g) (57).

Several mammalian cells, such as Chinese hamster ovary (CHO), HEK293, amniotic fluid CAP-T, and Vero cells, are used to produce VLPs (2, 14). These cells can produce complex and accurate post-translational modifications, and therefore, are preferred for the production of complex eVLPs containing multiple structural proteins. CHO cells are not human-derived; thus, compared with other mammalian cells, they have the advantage of a lower risk of contamination by human viruses. However, low yield (0.018–10 μg/mL) (14), difficulty in scale-up, and high cost are several factors that need attention. It has been shown that the CHO‐derived viral‐like particles for hepatitis B include a combination of glycosylated and non‐glycosylated HBsAg proteins, similar to those in patients’ sera. Examples of CHO‐based vaccines are GenHevac B^® and Sci‐B‐Vac™. These vaccines contain the HBsAg small and middle proteins (GenHevac B) or middle and large proteins (Sci‐B‐Vac) (58, 59). Similarly, hantavirus VLPs have been designed to co-express the nucleocapsid protein and glycoproteins (Gn and Gc) in CHO cells. These VLPs produce both humoral and cell-mediated immune responses against hantavirus (60). However, CHO-derived VLPs may be contaminated with HCPs. Certain HCPs, if not removed during subsequent purification processes, have been shown to induce immunogenic responses in patients. Others can shorten the shelf life of VLP vaccines via various mechanisms, including polysorbate degradation. Anti-CHO antibodies have been identified in 54% of individuals with no history of exposure to protein therapeutics (61). Therefore, a reduction in HCPs to minimal levels is required. While most HCPs are removed during downstream purification steps, certain ‘difficult-to-remove’ HCPs often remain (62). Additionally, stable transfection of mammalian cells is time-consuming, transient transfection has been used to produce VLPs in several studies. For example, Wu et al. demonstrated the effective production of influenza VLPs resembling original viruses in size, structure, glycosylation, and host factor composition (63). Similarly, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) VLPs was constructed by co-expressing four SARS-CoV-2 structural proteins in Vero E6 cells making it stable and unified, as compared to those from HEK293T cells.

Several VLP-based vaccine candidates tested in clinical and preclinical trials are summarized in Tables 5A , B , respectively. Several VLP-based vaccines have been developed against SARS-CoV-2, five of which are currently undergoing clinical trials. The main antigenic component of these vaccines is the spike protein, specifically the RBD, which is involved in viral entry and antibody neutralization. By displaying multiple full-length spike proteins or only RBDs on the VLPs surfaces, especially on eVLPs, these vaccines have shown high immunogenicity in clinical trials, generating neutralizing antibodies and inducing cell-mediated responses. However, introducing additional structural proteins as antigens in eVLP-based vaccines may lead to more complex and native-like structures and generate broad-spectrum immunity against various SARS-CoV-2 variants (3, 146).

Currently, only a few VLP-based vaccines are commercially available against various pathogens such as zika virus, HCV, HBV, and HPV due to various hurdles in their development processes. Therefore, these hurdles must be addressed to enhance commercialization. Researchers are trying to refine VLP-based vaccines at the formulation level by selecting appropriate adjuvants and stabilizers/protectants, administration routes, and delivery vehicles, such as liposomes, poly(lactide–co-glycolide) microparticles, or alginates to reduce the number of vaccine shots and dosage. Concomitantly, methods that can enhance the shelf life of vaccines and avoid cold chains are under investigation.

From an economic point of view, Gardasil is the most successful VLP-based vaccine, yielding over $48.7 billion in 2021. Gardasil has been replaced by Gardasil9 that is an adjuvanted non-infectious recombinant 9-valent vaccine. Gardasil9 has increased concentrations of L1 VLPs for HPV 16 and 18 in order to induce antibody responses in comparison to Gardasil (179). L1, is a ~55 kDa protein with the ability to spontaneously self-assemble into VLPs. In contrast, in other VLP based vaccines generated in the last three years, such as Cervarix for HPV and Engerix-B, Recombivax HB, and Hepavax-Gene for HBV, the revenue has not been reported. Cervarix contains purified proteins from two types of human papillomaviruses (types 16 and 18) (180). It is made using monophosporyl lipid A (MPL), a purified lipid (fat-like substance) extracted from bacteria, which enhances the immune response of the vaccine. VLPs and MPL are then fixed onto an aluminum compound to stimulate a better immune response. MPL is a heterogeneous mixture of congeneric lipid A with 4′-phophoryl groups and varying numbers of acyl chains (181). MPL adjuvant in the form of AS04 mimics a Toll-like receptor 4 agonist providing direct stimulation of antigen presenting cells, pronounced cellular and humoral immune responses, and long lasting antibody responses (179).The Hepavax-Gene (thiomersal free) and Engerix B are non-infectious recombinant hepatitis B vaccines that contain highly purified HBsAg. Engerix-B and Recombivax vaccines differ only in the concentration of HBsAg and the nature of the aluminum adjuvant. Engerix-B uses aluminum hydroxide as an adjuvant and trace amounts of thimerosal from the manufacturing process (182). On the other hand, Recombivax contains aluminum hydrophosphate sulfate. Currently, both academic and industry partners collaborate to produce cheaper vaccines that are affordable to low- and middle-income countries (LMIC). However, based on past events (e.g., HBV vaccination), this appears to not have occurred before. For example, life-saving vaccines such as the HPV VLPs Gardasil and Cervarix were first introduced at relatively high prices in industrialized countries and private sectors in developing countries. Therefore, reaching LMICs with these vaccines took longer than expected. The financial viability of biopharmaceutical companies is the main hurdle in this process and needs to be addressed. Commercial names of various licensed VLP vaccines are given in Table 6 .

PK, NA, KA, and RG were responsible for the conceptualization of the manuscript. SR, AJ, RR and RG: Contributed to latest literature review and manuscript drafting. All authors contributed to the article and approved the submitted version.