Platforms, advances, and technical challenges in virus-like particles-based vaccines

1Traditional 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. 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.

2VLPs 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.

3VLPs 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. 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. 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. 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.

4VLPs 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. 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. 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). Because VLPs elicit a protective response at lower doses, they can significantly reduce vaccine costs.

5Several 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. 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. 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. 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.

6This 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.

7VLP-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. All systems have their own advantages and drawbacks, which are briefly listed in Table 1.

8The 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. 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.

9Due 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. 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). 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.

10VLP 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. Plants have been used for VLP production of the Norwalk virus, HIV-1, 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 and the hepatitis A vaccine Epaxal.

11Three 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 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. This approach has been used to test a variety of VLPs, including HIV, chikungunya virus and Ebola. 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 and recombinant HBsAg. 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. There are many types of continuous modes such as chemostat, turbidostat, stressostat, and morbidostat. Chemostat is the most widely used continuous cultivation method. 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. 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. The corresponding yields from the different culture modes are summarized in Table 2.

12The 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. Each of the three culture modes have several advantages and disadvantages ( Table 3 ).

13Bacterial systems (mainly E. coli) are used to produce approximately 28% of all VLPs. 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. 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. 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. Subsequently, a glycoengineered E. coli strain, CLM24, was developed to express both Cj PglB OTase and Cj lipid-linked oligosaccharides. 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. 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. 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.

14Insect, yeast, and mammalian platforms have also been used for the production of VLPs since the 1980s. 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. For example, the secretory form of influenza VLPs consisting of hemagglutinin (HA) and matrix proteins was successfully produced by recombinant insect cells. 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. 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. H. polymorpha has several unique characteristics such as thermostability, avoidance of hyperglycosylation, and use of several carbon sources. This platform has been established for the cost-effective production of VLPs at a larger scale (40 mg/100 g).

15Several mammalian cells, such as Chinese hamster ovary (CHO), HEK293, amniotic fluid CAP-T, and Vero cells, are used to produce VLPs. 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), 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). 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. 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. 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. 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. 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.

16Cell concentration at infection (CCI), multiplicity of infection (MOI), and time of harvest (TOH) in the baculovirus system strongly influence protein expression and, consequently, assembly of VLP. 72–120 h post-infection is considered the optimal time of harvest. Outside this period, various parameters can hinder productivity, such as cell lysis, protein degradation, and difficulties in downstream processing owing to the presence of contaminant proteins, hosts, or viral DNA. Incomplete VLP may also be released into the medium along with target mature VLP and can hinder the downstream processing of VLP. Differences in volumetric productivity, either due to low or high MOI, can be counterbalanced by modulating CCI. However, CCI is inversely correlated with productivity. Although perfusion or fed-batch techniques may overcome these limitations, they are neither practical nor economically feasible. Therefore, the development of novel techniques for process optimization is required.

17The assembly of VLPs involves assembling single or multiple structural capsid proteins in appropriate production hosts (in-vivo assembly) or in cell-free conditions (in-vitro assembly). The nature of the stabilizing contacts can influence the VLP assembly route. Protein-protein interactions protect the viral genetic material from the degradation and facilitate the establishment of a relatively stable carrier/delivery system. Similarly, viral genomes coevolution with their own structural elements has resulted in interactions in which nucleic acids are the primary driving mechanism for VLP formation. For example, the formation of HCV VLP is dependent on the interaction of highly basic N-terminal 120or 124 amino acids of the core protein with a 5′ untranslated region containing expected secondary structural elements. The assembly and disassembly of VLPs may also depend on the ionic or reducing conditions in which they are assembled. In more complex viruses, scaffolding proteins play an essential structural role in ensuring correct assembly of large viruses. For example, P22a is a critical scaffolding protein in the particle assembly of the herpes simplex virus. Assembly is not usually a single high‐order reaction, but can sometimes include a cascade of low‐order intermediate reactions. These intermediate reactions at various phases of assembly can be examined or trapped by mutagenesis of structural proteins and their expression in a suitable system. The uniformity and self-assembly of VLPs, along with their ability to withstand chemical modifications primarily on the outer surface, make them flexible and stable alternatives to nanoparticles, such as liposomes and metal assemblies.

18Self-assembly of VLPs is driven by interactions between capsid proteins and often with the viral genome. For example, approximately 25% of the HBsAg monomers in the RTS,S (Mosquirix™; the first vaccine for malaria) are genetically fused to the truncated circumsporozoite protein (CSP) and serve as protein carriers. The region of the CSP included in the RTS,S vaccine includes immunodominant B-cell epitopes (last 18 four amino acids (NANP) repeats) and three known T-cell epitopes: a highly variable CD4+ T-cell epitope before the Thrombospondin (TSP)-like domain, a highly variable CD8+ T-cell epitope within the TSP-like domain, and a conserved “universal” CD4+ T cell epitope at the C-terminus. These epitopes of Mosquirix enhanced antigen presentation to the immune system and likely facilitated strong anti-CSP antibody and T-cell responses measured in vaccinated individuals. Mosquirix has received approval from the regulatory authorities.

19To obtain superior assembly effects, cell-free or in vitro assembly of VLPs requires control of variables such as temperature, pH, and specific assembly solution. The assembly of VLPs in vivo necessitates a suitable thermodynamic environment for the host, which might result in the formation of malformed structures and pose technical challenges in VLP assembly and production. In this context, thermodynamic studies may be useful for identifying the most suitable environmental conditions for production during upstream processing as well as for maintaining stability throughout downstream processing (DSP) and storage. Various studies have demonstrated the effects of several physicochemical parameters (pH, ionic strength, temperature, and correct stoichiometric ratio) on the formation of VLPs. When optimal conditions are not available, VLP destruction increases considerably. Additionally, owing to their large structure, VLPs may occasionally lose their self-assembling property or cause protein misfolding, resulting in the formation of defective VLPs. Time is another factor that renders antigen assembly laborious. Therefore, optimization is essential for appropriate VLP assembly and should be investigated further.

20Several process parameters, such as dissolved oxygen concentration, pH, temperature, agitation rate, inlet gas flow, and composition, influence VLP production and pose technical challenges either by hindering the cell growth or metabolic state of the cells or by interfering with the mechanisms responsible for post-translational modification. Limited or excess oxygen induces proteases that can degrade the product of interest. When oxygen-derived free radicals are formed owing to a high concentration of dissolved oxygen, they either induce oxidative stress in cells or cause oxidative damage to proteins. Temperature is another parameter that can control oxygen solubility.

21The use of headspace aeration (bubble-free systems) or nonionic copolymers may provide a solution depending on the degree of sensitivity of the cells. Cellular damage can be eliminated in a bubble-free system, but productivity decreases due to inadequate levels of dissolved oxygen. However, nonionic copolymers do not interfere with dissolved oxygen. These copolymers lower the surface tension of the culture medium and increase cell membrane rigidity, thereby inhibiting the attachment of cells to bubbles and increasing cell resistance to hydrodynamic forces.

22The separation of a complex mixture of proteins from VLP using transduction is challenging because of their similar size and molecular weight. The same problem does not exist in the case of transfection, which thus, significantly decreases the complexity of downstream processing. Serum-free medium further improves or facilitates downstream processing because it does not contain any animal-derived supplements. Controlling various process conditions, such as optimal salt, pH, and ion concentrations, can reduce or avoid protein aggregation, which is a major challenge in downstream processing. However, aggregation during large-scale production of VLPs, owing to their larger size, leads to the loss of a considerable portion of the target protein or reduced immunogenicity. In a recent study, a new membrane chromatography procedure was developed to produce nano-VLPs against Ebola. This method reduces VLP size to a more manageable range, where it can not only retain its temperature stability and antigen structure, but also stimulate immunity in mice. In addition, lyophilization can be used to increase its thermostability. Lyophilization and freeze-drying are used worldwide. However, they are especially effective for tropical countries that do not have a reliable “cold-chain” of refrigeration. A patent was filed for the methodology against the Ebola virus in 2015 (US20170274063A1); however, in 2017, it was abandoned. Tangential flow filtration, gel permeation, ion exchange, size exclusion chromatography, and mixed-mode chromatography, including the use of improved disposable membrane technologies, can improve the productivity of purified VLP.

23The biochemical characterization of VLPs is usually performed using various techniques such as sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis, western blotting (WB), enzyme-linked immunoassay (ELISA), and bicinchoninic acid (BCA) protein quantification assays. Flow cytometry is now being exploited as a tool for the study and validation of VLPs. However, these techniques cannot differentiate between unassembled and partially or completely assembled VLPs and are time-consuming, have low sensitivity, and require high sample volumes. This leads to either the over-or underestimation of proteins, compromising downstream processing. Some of the issues mentioned above have been effectively visualized and addressed using Transmission electron microscopy (TEM). Particle size analysis using light-scattering techniques is commonly adopted to measure the hydrodynamic size and homogeneity (polydispersity) of VLPs in solution. Additionally, orthogonal methods, such as high-performance liquid chromatography (HPLC), SDS-capillary gel electrophoresis, MALDI-TOF MS, and capillary zone electrophoresis, have been developed to overcome the limitations posed by SDS-PAGE, WB, ELISA etc. Two-dimensional fluorometry is an optical fiber-based technology that can monitor several compounds, either present outside or inside cells. Therefore, in situ monitoring of the kinetics of VLPs is possible using this technology.

24Critical Quality Attributes (CQAs), such as the biophysical, biochemical, structural, and immunochemical properties of VLPs, are critical parameters in developing vaccine candidates with good stability and delivering consistent products for the preclinical and clinical stages. In addition, these parameters also help in post-licensure life-cycle management to support market demand by upgrading the process or scale-up.

25Various methods are used to characterize the physical and structural attributes of VLPs, such as transmission electron microscopy (TEM), Atomic force microscopy (AFM) for morphology, size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) for monodispersity, dynamic light scattering (DLS), cloud point, surface plasmon resonance (SPR) to check propensity of aggregation and circular dichroism (CD) and differential scanning calorimetry (DSC) to check thermal stability. However, it should be noted that drying of samples is required during TEM or AFM, which can introduce artifacts caused by the dehydration of VLPs. Cryo-EM can overcome the drawbacks of TEM and AFM because it preserves the VLP structure in its native form. However, cryo-EM is only useful if the VLP preparation is sufficiently homogeneous, although a pleomorphic nature can also be observed. Similarly, the AFM in the solution also reduced the drying artifacts. Another major challenge in VLP characterization is accurate measurement or quantification of VLPs to derive an accurate dose. Nanoparticle tracking analysis (NTA)/tunable resistive pulse sensing (TRPS), electrospray-differential mobility analysis (ES-DMA) and asymmetric flow field flow fractionation with multi-angle light scattering detection (AF4-MALS) have been used to accurately measure the size distribution accurately. These methods are cost-efficient compared to TEM and are used to quantitatively monitor batch consistency for new vaccines.

26Activity-related attributes are also important for checking the structure–function relationship in VLP-based vaccines or the integrity of key epitopes. Tools such as bilayer interferometry (BLI) and SPR are used to check epitope-specific antigenicity and epitope mapping. In vitro relative potency or mouse potency assays are used to test the release and stability of VLPs. These assays are required to define the quality and quantity of epitopes on recombinant antigens. Therefore, they play a major role in the implementation of process improvements or scale-up.

27Additionally, expressing VLPs in a suitable host to provide an appropriate environment and post-purification disassembly/reassembly are essential for enhancing the stability, homogeneity, and immunogenicity of VLPs. For example, HBsAg particles contain 30% lipid by mass; therefore, a suitable host that can provide an appropriate lipid composition should be chosen. Similarly, HPV VLPs derived from E. coli are heterogeneous in nature because of the many ill-formed or incomplete particles. This heterogeneity can be reduced by post-purification disassembly followed by reassembly, as reported previously.

28Demonstration and maintenance of comparability between different lots of preclinical/clinical-grade materials and between small-scale preclinical and large-scale clinical trial materials is a regulatory requirement. Process changes are implemented based on the lot-to-lot comparability exercise, an important component of chemistry, manufacturing, and control (CMC) batch analysis. Lot-to-lot comparability relies on the physical and functional properties of the vaccine, which are responsible for predicting the consistency of batches across different lots. This analysis ensures that vaccine lots are equivalent in potency, purity, and physicochemical integrity. Any process modification and formulation change between these phases must also be verified by CQA-based comparability, which is required for Good Manufacturing Practice (GMP).

29Chimeric VLPs are produced by assembling the structural proteins or epitopes of different viruses. For example, in the M2-HPV model, the M2e influenza epitope was inserted into the HPV 16 L1 sequence in Nicotiana benthamiana plants. L1 is the major capsid protein of HPV. The production process for chimeric VLPs depends on various factors, such as glycosylation efficiency, intermolecular chemical bonding, steric hindrance, and cell type; therefore, it is highly unpredictable. This unpredictability may lead to the formation of heterogeneous VLPs. Adding nonpeptide antigens to chimeric VLPs is another challenge because introduced antigens cannot adopt a quaternary structure and thus lead to inappropriate folding of the protein. These epitopes are not optimally exposed; therefore, the immune responses obtained against such epitopes are often limited. Thus, a VLP pseudotyping strategy or modular molecular assembly approach is a viable solution for overcoming this drawback. In the first step of the two-step process, native VLPs and target antigens are synthesized separately. In the second step, in vitro assembly of the two components is performed by either covalent or noncovalent binding, which links the target antigen to the surface of the preassembled VLP. This approach significantly enhances the combination of a full-length correctly folded target protein with VLPs. Chemical cross-linking is the most commonly used method to facilitate the binding of antigens and native VLPs. Two examples of chemical crosslinking are the M2-HPV VLP conjugate vaccines of Merck & Co, Inc. and Cytos Biotechnology AG, where the M2 peptide of influenza A is conjugated to VLP. Another example is the chemical coupling of the receptor-binding domain (RBD) of SARS-CoV-2 to cucumber mosaic virus derived virus-like particles (CuMVTTVLP) by a Swiss biotech company, Saiba-AG. These CuMVTT VLPs incorporate a universal T-cell epitope derived from the tetanus toxin. In addition, these VLPs package bacterial RNA, which is a ligand for Toll-like receptor (TLR) 7/8 and serves as a potent adjuvant by engaging these innate receptors in specific B cells.

30VLPs can become very unstable and tend to phase separately in solution. The stability of VLPs is essential for producing viable antigens for VLP-based vaccine candidates. This is only possible after obtaining a stable formulation of VLPs. Therefore, preserving critical epitopes on VLPs, along with immunogenicity, is equally important. The introduction of nonionic surfactants into HPV VLP aqueous solutions significantly enhanced their stabilization and reduced aggregation due to heat stress and physical agitation. Generally, eVLPs are more susceptible to external environmental conditions than non-enveloped VLPs because of various factors, such as temperature changes, shear stress, fluid dynamics, agitation rate, chemical treatment, and dissolved oxygen, all of which affect the integrity and stability of VLP particles. Several modifications have been made to improve the thermostability of the eVLPs. A typical procedure involves insertion of stabilizing mutations. A study discovered that inserting a stabilizing mutation in the coat proteins of poliovirus type 3 VLPs increases their stability when compared to wild-type VLPs. Because adjuvants are primarily used to enhance the stability of VLPs, proper dissolution conditions for the removal of adjuvants are also required for morphological and antigenicity analysis of VLPs. In-vitro relative potency (IVRP) or mouse potency under proper dissolution conditions to fully recover the antigen is used to assess the stability of vaccines over many months.

31Another important parameter is to define the stability of VLPs in three storage conditions: real-time (long-term), accelerated, and stress (high-temperature) conditions. Long-term stability studies define the conditions needed for vaccine preservation and expiration date. Accelerated and stress condition tests are used to define the stability of the vaccine in the short-term deviation from preservation and extreme conditions, respectively. However, defining stability under these conditions is challenging because a conceptualized matrix is not available for VLPs owing to their non-linear nature in response to thermal and other external factors and may be very specific to the nature and composites of VLP. The stability tests include identity, appearance, volume of fill, pH, osmolarity, adjuvant content, detergent content, sterility test, endotoxin, impact on potency, and toxicity.

32Similarly, to avoid the freezing–de-freezing cycle, critical attention should be paid to choosing appropriate and compatible cryoprotectants to preserve the native structure of VLPs.

33Several purification steps, such as ion exchange, size-exclusion chromatography, and ultracentrifugation, are used to obtain purified VLPs. Furthermore, HCP are removed by polishing. This step is followed by sterile filtration and formulation to obtain a safe and effective vaccine. At the industrial scale, size-exclusion chromatographic purification or sucrose gradient ultracentrifugation steps are used, which increase production costs. Many scientists are working on replacing these steps with more robust purification steps. For example, a robust purification protocol obtained approximately 90% pure foot and mouth disease virus (FMDV) VLPs. This protocol does not include non-preferred industrial size-exclusion chromatographic purification or sucrose gradient ultracentrifugation steps. This approach could be used for large-scale industrial production of E. coli-based VLPs against FMDV infections of different serotypes, making this approach cost-effective. Carvalho et al. showed that the use of sulfated cellulose membrane adsorbers (SCMA) enhances the scalability of vaccines compared to conventional ion exchanger membrane adsorbers by reducing the number of purification steps. This study suggested that SCMA could qualify as a generic platform for purifying VLP-based influenza vaccines. However, if the same techniques can be used to produce several different VLPs, it has not been tested and needs further investigation.

34Similarly, a chromatographic procedure was used to purify HIV-1 gag VLPs using a mammalian platform, in which the clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. Gag, or group-specific antigen, is the major structural protein of HIV-1 and all other retroviruses, and comprises approximately 50% of the mass of a viral particle. This method showed a 220-fold improvement in productivity compared to density gradient centrifugation. Plants such as tobacco and related Nicotiana species are commonly used to generate plant-made pharmaceutical proteins (PMPs). However, these plants have high levels of phenolics and toxic alkaloids; therefore, the downstream processing of target PMPs is hindered by technical and regulatory difficulties. Hence, lettuce-based production systems are used instead of tobacco-based platforms because of their low levels of secondary metabolites. More complex VLPs have production challenges owing to the incorporation of envelope proteins or their stable nature. Therefore, new technologies must be developed. For example, in the case of enveloped HIV VLP, crossflow filtration across a hollow-fiber cartridge membrane under low-stress conditions concentrates the desired material with 95% recovery.

35VLPs have intrinsic immunostimulatory properties because of their ability to activate antigen-presenting cells directly and therefore have great potential for use as antigenic platforms. This immunogenicity can be further improved and oriented by incorporating non-coding RNAs (ncRNAs) that act as TLR7/8 ligands. For example, the incorporation of ncRNA into murine leukemia virus (MLV)-derived VLPs provides further TLR-mediated activation and Th1-type CD4+ and CD8+ T-cell response orientation. TLRs are expressed by innate immune cells, predominantly antigen-presenting cells (APCs), and recognize various pathogen components. Upon binding to PAMPs, TLR signaling activates APCs, which leads to the production of cytokines, increased co-stimulatory molecule expression, and an enhanced capacity to present and cross-present antigens on MHC class I molecules.

36The expression of antigens either in their core or on the surface of VLPs significantly improves their immunogenicity, favoring the induction of both B and T-cell-mediated immune responses. Compared to non-enveloped VLPs, eVLPs have the added advantage of presenting the surface proteins of enveloped viruses in their native form. These features can enhance both the humoral and cell-mediated immune responses. For example, the gag protein of Moloney MLV in HEK-293 cells was cleaved to generate a group of proteins that function to create the structured inner core of a VLP. These include matrix, capsid, and nucleocapsid proteins. The capsid protein is the main structural element of a mature virus particle, forming the core-shell around the nucleocapsid-RNA complex, whereas the matrix protein remains linked to the viral lipid bilayer. The commercial name of this vaccine is VBI-1501, which showed high immunogenicity in animals and was used in a phase I clinical trial. Another eVLP vaccine candidate, VBI-1901, is undergoing phase II testing for therapeutic immunization against glioblastoma. Similarly, two other vaccines, VBI-2902a and VBI-2905a, are eVLP-based COVID-19 vaccines for Wuhan and B.1.351 (Beta) variants of SARS-CoV-2, respectively. These vaccines contain a stabilized form of the spike protein of SARS-CoV-2 inserted into the eVLP lipid bilayer.

37The immunogenicity of VLPs can be increased by engineering VLPs, allowing for a more versatile display of antigens. To maximize versatility in displaying foreign antigens of varied compositions, sizes, and structures, the recombinant fusion approach is adopted. In this approach, foreign antigens are inserted into sites inside the viral structural protein to display them on the surface of the resulting VLP’s. However, because peptide insertions into viral capsid proteins frequently result in protein-folding failures, peptide insertions frequently impact the ability of recombinant proteins to assemble into VLPs. To avoid these failures, preformed VLPs might be employed as scaffolding. Foreign antigens are attached to the VLP scaffold using various conjugation techniques. Similarly, the magnitude and longevity of immune responses of VLPs can also be enhanced by modifying VLPs in such a way that their interactions with specific receptors on or within immune cells, interactions with molecules that regulate trafficking, and compatibility with adjuvants can be increased. Many VLPs have molecular and structural properties that can spontaneously stimulate the immune system without adjuvants. Nevertheless, the use of adjuvants in VLPs vaccine formulations may enhance immunogenicity and stimulate a specific immune response.

38Table 4 summarizes the most recent methods for increasing VLP productivity/immunogenicity.

39Several 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.

40Currently, 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.

41From 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. 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). 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. 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.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. 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.

42PK, 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.