These VLPs are often made up of single or many self-assembled components of the targeted pathogen or viral protein structures. There is no host cell membrane (lipid envelope) in these newly formed VLPs. The expression of the major viral nucleocapsid proteins is primarily responsible for the formation of these VLPs. Non-enveloped VLPs are still being investigated as preferable candidates for developing subunit vaccines against several pathogenic diseases as they are easier to produce and purify (Naskalska and Pyrć, 2015). Furthermore, these VLPs are smaller in size that allows them to easily cross the tissue barrier and drain to lymph nodes (Keikha et al., 2021). The major structural component of the virion is formed by the single, virally encoded protein and is derived from pathogens, such as caliciviruses, papillomaviruses, and parvoviruses (Mohsen et al., 2017), whereas multi-protein non-enveloped VLPs are much more complex containing multiple interacting capsid proteins. They show several striking structural characteristics, such as many complicated concentric layers of different capsid proteins. They are more difficult to make than those that are made up of only one main capsid protein (CP) and derived from infectious bursal disease virus, poliovirus, and retroviruses (Lua et al., 2014).

In comparison to non-enveloped VLPs, enveloped VLPs (eVLPs) have a far more complicated composition. These nano-structures consist of a cell membrane acquired from the host cell called an envelope, with viral proteins present on the outer surface (Dai et al., 2018). One or more than one glycoprotein spikes are embedded in their lipid bilayers and act as a target antigen for producing neutralizing antibodies. These eVLPs show a higher degree of flexibility as they target antigenic epitopes from the same or heterologous viruses. For example, the eVLPs have been developed that contain Gag protein from SIV and Env protein from HIV (Kushnir et al., 2012). Although it may affect downstream applications because of the presence of the host protein. The eVLPs have been used to develop vaccines against viral diseases, such as the hantaan virus, hepatitis C virus (HCV), influenza A, and retroviruses (Wetzel et al., 2019a). These large eVLPs have a size greater than 100 nm, so there is a chance that they might aggregate at the site of injection and will not reach the lymph nodes that limit their application (Keikha et al., 2021). In comparison, lipid nanoparticles carrying mRNA vaccines have a size range of 100 nm or less and show high efficacy in delivering vaccines to the targeted site (Editorial., 2021; Żak and Zangi, 2021).

Chimeric virus-like particles (cVLPs) are considered an effective tool for developing vaccines that provide broader, more powerful, and comprehensive protection against emerging infectious diseases (Wetzel et al., 2019a). The complex, multi-protein macrostructures contain epitopes of different viruses (Latham and Galarza, 2001). cVLPs can be created by constructing recombinant DNA molecules that encode both the relevant viral protein and a foreign peptide or protein. These genetically engineered cVLPs display a high number of repetitive sequences on their surface that can be loaded with exogenous antigens from other viruses via chemical conjugation or genetic fusion (Caldeira et al., 2020). Different chimeric vaccines have undergone clinical trials, including the VLP-based vaccine (M2–HBcAg) against hepatitis, an antimalarial vaccine (MalariVax) (Nooraei et al., 2021), anti-influenza A, anti-HIV (Rutgers et al., 1988), and the nicotine-Qb VLP vaccine to reduced nicotine level in the blood of smokers (Maurer et al., 2005). These chimeric particles are advantageous as they substantially increase immune response and antibody titer in response to foreign antigens. Following administration of cVLPs, they induce strong cytolytic T lymphocyte immune responses (Qian et al., 2020). These vaccines are also targeted against non-infectious diseases, such as hypertension, Alzheimer’s, nicotine addiction, allergies, and diabetes (Huang et al., 2017). The upstream and downstream processing yield of cVLPs is usually low, and their in vivo stability is also quite uncertain (Buonaguro and Buonaguro, 2014).

VLPs are generally considered more stable than subunit vaccines (Dai et al., 2018). However, when the environmental conditions change, especially during downstream processing, VLPs can become very unstable as they lack the genetic material of the virus. Despite the fact that multiple VLP vaccines are already available in the marketplace, some of the candidates’ vaccines have stability issues (Mohsen et al., 2018). Generally, eVLPs are often more susceptible to external environmental conditions as compared to non-enveloped VLPs (Dai et al., 2018). Variations in conditions such as a change in temperature, shear stress, dissolved oxygen, fluid dynamics, agitation rate, and chemical treatment may all have an impact on the particle’s integrity and stability (Roldao et al., 2011). Moreover, this structural breakdown significantly reduces the immunogenicity of eVLPs. It also interferes with cell growth and the production of metabolic proteins, which has an impact the VLP production. It has been one of the key obstacles to using them as an alternative for a live virus in vaccine manufacture. However, several modifications have been made to increase their thermostability. One typical procedure is the insertion of stabilizing mutations (Dai et al., 2018). A study conducted by Marsian et al. (2017) showed that when stabilizing mutation was induced within the coat proteins of poliovirus type 3 VLP, it become more stable than wild type VLPs. It modified capsid precursor and the viral protease without disturbing its antigenic epitopes and structural confirmation. In another study stability of chimeric (HBcAg-VLP) was increased by the addition of C-terminal linker-hexahistidine-peptide (Schumacher et al., 2018).

Some VLP-based vaccines are much more complex and, as a result, have a higher cost of production. The numerous impurities associated with eVLPs pose a daunting challenge. During downstream processing (DSP) various impurities like host cell debris (HCD), host cell protein (HCP), and host cell DNA (HCD) are co-purified as in the case of enveloped baculovirus particles. These contaminations can cause undesirable side effects in vaccines if not removed properly during DSP (van Oers, 2011). Large scale production and purification of VLPs require different processes like density gradients or even chromatography to make the final formulated product. These complex processes are very costly and time-consuming (Wetzel et al., 2019a). This also leads to difficulty in industrial scale production and requires several quality control efforts as various downstream processing steps may worsen the VLPs quality (Diamos et al., 2019). A more robust and better analytical methods are required to ensure the quality and quantity of the product that can facilitate their clinical and pharmaceutical utilization (Moleirinho et al., 2020). Different strategies such as clone screening, high-throughput screening, bioreactor engineering, material/matrix screening, filtration, flow-through or size-exclusion chromatography, and polishing have been implemented during upstream and downstream processing for scalable and cost-effective industrial manufacturing of VLPs (Vicente et al., 2011; Lagoutte et al., 2016).

The genetic fusion of the sequences of epitope into VLPs can sometimes be challenging, as VLPs may lose their self-assembly property or cause particle misfolding (Guo et al., 2019). Genetically fusion of antigen to capsid protein of virus often hinders either antigen assembly or VLP, making the technique laborious. Therefore, time-intensive planning and optimization are required for individually testing every single antigen (Lampinen et al., 2021).

Most of the bacterial systems are focused on well-studied industrial strains and expression vectors of Escherichia coli (Zeltins, 2013). Bacterial cell cultures were investigated as a platform for VLP development, with benefits in terms of cost and scalability (Kim et al., 2014). The other beneficial features of using bacterial systems for VLP production include (a) easy manipulation, (b) high-level expression, (c) fast growth rate, (d) genetic stability, and (e) simplicity of expression (Masavuli et al., 2017).

Huo et al. (2018) used pCold expression vector to express and purify norovirus (NoV) VLPs in E. coli strain (BL21) and demonstrated the similar binding pattern for VLPs assembled in E. coli as for Sf9 cells assembled VLPs. Similarly, in a recent study, the full-length CP of type 2 porcine circovirus and VP2 protein of porcine parvovirus were expressed in E. coli, which were self-assembled into VLPs. The study suggested that the expression of the CP and VP2 in E. coli is possible for the mass development of VLP vaccines (Liu et al., 2020). Yazdani et al. (2019) investigated the possibility of utilizing VLPs of grapevine fanleaf virus (GFLV) as a potential vector for presenting the L2 epitope of HPV. The antigenic determinant sequence was incorporated genetically into the GFLV capsid protein’s “αB-αB” domain C, which was then overexpressed in E. coli and Pichia pastoris. In E. coli, the highest expression yield was observed. For Hepatitis E virus (HEV), the ORF2 protein region 368–606 aa was purified in vitro from the insoluble E. coli fraction that is assembled into VLPs. This HEV VLP promises 100% effectiveness in clinical trials against symptomatic HEV and is approved as a vaccine for commercial use in China (Gupta et al., 2020). In another study, infectious hypodermal and hematopoietic necrosis virus (IHHNV) VLPs from CP of recombinant IHHNV were reconstructed in E. coli and showed excellent physical stability (Kiatmetha et al., 2018).

Bacterial systems are not always the optimal VLP development strategy because of several factors, including (a) poor immunogenicity, (b) inability to develop recombinant proteins with mammalian−like post-translational modifications (PTMs), (c) issues of protein solubility, (d) inability to create the correct disulfide bonds, and (e) presence of bacterial endotoxins/or lipopolysaccharides in preparation of recombinant proteins (Shirbaghaee and Bolhassani, 2016; Masavuli et al., 2017).

Brito and Singh (2011) have highlighted the acceptable level of endotoxins for various types of vaccines. There are various methods by which endotoxins are removed during the purification step, such as immobilized sepharose, surfactants, activated carbon, ultrafiltration, and anion exchange chromatography. However, the use of these techniques often leads to a considerable reduction in yield, an increase in cost, or a lack in the biological activity of the target protein. Therefore, ClearColi™, a genetically engineered E. coli strain with a genetically modified LPS which does not elicit an endotoxic reaction in humans, has been developed (Mamat et al., 2013).

Eukaryotic ESs are a compelling alternative to prokaryotic ones, particularly when it comes to addressing the issue of bacterial endotoxins in vaccine production (Shirbaghaee and Bolhassani, 2016). VLPs can be produced more cost-effectively by recombinant expression of proteins in yeast cells. Manipulation of genes in yeasts is comparatively straightforward, and the transformed cells can grow to extremely high densities until the expression of recombinant proteins is induced. This allows commercial-scale fermenters to produce VLPs in large volumes (Stephen et al., 2018). Some mammalian viruses’ structural genes expressed in yeast can form the VLPs (Shirbaghaee and Bolhassani, 2016).

The FDA has approved some yeast-derived VLP vaccines, including Gardasil^® and Gardasil9^® against HPV and Mosquirix™ against P. falciparum (Nooraei et al., 2021). Some studies have investigated VLP production using yeast as an ES. Wetzel et al. (2018) established a robust and cost-effective yeast model for the production of cVLPs. The duck HBV membrane integral small surface protein (dS) was selected as the scaffold for VLP, and the safe and industrially applied Hansenula polymorpha yeast as the heterologous expression host. 8 distinct antigens of high molecular weight were derived from four viruses that infect animals and are genetically linked to a protein dS, and then the recombinant isolates were identified and purified. The fusion proteins were highly expressed in all cases, and it was possible to generate chimeric VLP comprising both proteins after co-production with protein dS. The production system based on yeast allows for a low-cost product that is not restricted to small-scale basic research. In another study, Alireza et al. (2018) produced chimeric protein L1/L2 VLPs in the P. Pastoris system by first inserting a cross-neutralizing epitope from the gene HPV-16 L2 into the gene L1 HPV-16. Following that, the chimeric L1/L2 HPV-16 had been introduced into the plasmid (pPICZA) and expressed in P. pastoris. The ELISA results for L1-HPV-16 Ab as well as L2-HPV-16 Ab detection indicated a positive reaction with sensitivity comparable to the commercial testing kit. Similarly, in the P. pastoris ES, Gupta et al. (2020) produced a recombinant VLP against HEV that included 112–608 aa region of the protein ORF2. The results showed that for the development of 112–608 aa VLP, the P. pastoris ES seems to be a superior and safer alternative to the baculovirus (Bv) ES.

During viral infection, enteroviruses like poliovirus, generate empty capsids that are antigenically indistinguishable from that of mature virions. The recombinant synthesis of such capsids with the help of heterologous systems like yeast has enormous potential as candidates for the VLP vaccine. Sherry et al. (2020) showed VLP production in P. pastoris through co-expression of the viral protease 3CD and the structural precursor protein P1.

Construction of yeast ESs is much more challenging than bacterial ESs, especially the Pichia and Hansenula strains. Furthermore, the VLP yield is lower than that of E. coli. Another disadvantage of the yeast ES is its lack of resemblance to mammalian ESs in protein PTMs, particularly glycosylation (Shirbaghaee and Bolhassani, 2016). Their glycoforms are mostly of the high type of mannose, which is undesirable for most pharmaceutical glycoproteins (GPs) (Kim et al., 2014).

Baculovirus-based protein expression in insect cell lines has appeared as an effective tool for developing complicated protein-based biologics for a variety of purposes, extending from multiprotein complexes to the development of proteins for therapeutic use, such as VLPs (Sari-Ak et al., 2019). Bv is unusual among widely used viral vectors in its ability to tolerate heterologous DNA in large amounts and deliver it faithfully to the desired host cell (Gupta et al., 2019). Gopal and Schneemann (2018) described detailed procedures for producing recombinant baculoviruses (rBVs), screening for VLP expression in insect cell lines, and purifying VLPs.

The Bv-insect cell system is a two-step procedure in which insect cells are originally grown to the required cell concentration before being infected with rBVs for protein expression (Roldão et al., 2010). Recombinant protein and VLP production using Bv expression vector systems (EVSs) is fast, versatile, and provide substantial yields (Strobl et al., 2020). This system can produce exceptionally high protein production levels, combined with complex eukaryotic protein PTMs, which may be crucial for the proper self-assembly and release of some VLPs (Chaves et al., 2018).

MultiBac is an innovative Bv EVS that comprises of a viral genome that has been engineered to suit specific purposes. Recently, a team of scientists described the development of a MultiBac-based VLP-factory, dependent on the CP (M1) of influenza virus and its utilization in generating an arsenal of influenza-derived VLPs with functional modifications in influenza virus hemagglutinin (HA), which are expected to regulate the VLP-derived immunological response (Sari-Ak et al., 2019).

The insect cell-Bv EVSs have proven to be as effective as conventional egg- and cell-based approaches in influenza virus vaccine production with additional features like high production yields, and short production times. Moreover, rBVs construction has become faster, easier, and more flexible, allowing for the fusion of genes from different types and/or subtypes of influenza viruses inside the same expression vector. Sequeira et al. (2018) successfully created a robust High Five cell-based insect platform that combines stable expression with Bv-mediated expression to generate multivalent influenza VLPs. The capability of this modular approach has been proven by infecting the High-Five cell line with 2 distinct HA proteins of subtype H3 (called HA2 and HA1) with a Bv expressing M1 and 3 additional HA proteins of subtype H3 (called HA5, HA4, and HA3), to generate pentavalent VLPs (H3).

The Zaire Ebola virus serotype (ZEBOV) is the most virulent and has the highest mortality rates among other serotypes. ZEBOV-VLPs development have been achieved in insect and mammalian cell lines via co-expression of 3 viral structural proteins, the nucleocapsid protein (NP), the matrix structural protein (VP40), and the glycoprotein (GP). A technique for generating ZEBOV-VLPs in insect cell line was reported by Pastor et al. (2019), which basically consists of employing a high multiplicity of infection (MOI) of bac-GP and bac-VP40, and limiting the NP expression, either via preventing infection or by lowering the bac-NP MOI, was the most suitable for developing VLP.

The main limitations of the insect cell ES are (a) protein contamination by enveloped baculoviruses, (b) difficult to scale-up, and (c) simpler N-glycosylation than mammalian cells (Shirbaghaee and Bolhassani, 2016; Masavuli et al., 2017).

For more than two decades, various mammalian cell lines such as murine myeloma (Sp2/0, NS0), Chinese hamster ovary (CHO), murine C127, baby hamster kidney (BHK21), HT-1080, and HEK293 were established as a possible source of commercialized protein therapeutics for medical purposes, due to their capability to properly fold, assemble, and post-translational modification to proteins (Dumont et al., 2016; Shirbaghaee and Bolhassani, 2016). Systems based on mammalian cell culture offer many benefits, including consistency and flexibility during the development process. It also helps glycosylated proteins to be recovered with compositions of lipid membrane similar to the virus’s host. It has been reported that the stable transfection of viral genes into mammalian cell lines, including 293 or Vero cells, results in VLP production (Buffin et al., 2019).

Several studies reported the efficient production of VLPs from mammalian cell cultures. Hsin et al. (2018) developed a MERS VLP system using Huh7 cells as an expression system for understanding virus infection and morphogenesis. In another study, influenza VLPs encoding neuraminidase (NA), hemagglutinin (HA), and matrix M1 proteins had been expressed in Vero, 293 T, or CHO-K1 cell lines, using transient transfection. Preclinical studies in BALB/c mice revealed that influenza VLPs, when given intramuscularly, were significantly immunogenic at low dosages, inducing functional Abs against NA and HA (Buffin et al., 2019). Wu et al. (2010) designed a scalable method for the effective production of various subtypes of influenza VLPs expressed in mammalian cell line. The study demonstrated that these mammalian influenza VLPs were very similar to the original viruses in particle size, structure, host factor composition, and viral antigen glycosylation. Similarly, in another work, an inducible cell line of human embryonic kidney HEK-293 expressing NA and HA was developed and utilized to generate VLPs following transient transfection with a plasmid encoding HIV-1 Gag (Venereo-Sanchez et al., 2017). A protocol developed for the synthesis of HIV-1 Gag VLP in mammalian cell suspension cultures via transient gene expression showed that the large proportion of Gag-GFPs present in the supernatants of cell culture was fully assembled into VLPs of the predicted morphology and size consistent with immature particles of HIV-1 (Cervera et al., 2013).

Expression systems based on mammalian cells require large-scale production facilities, like fermentation bioreactors, which are prohibitively expensive to construct. As a result, the high cost of production is a challenging part of the cell-based mammalian ES (Kim et al., 2014). The other limitations include lengthy-expression time, low yield, and vulnerability to infections with mammalian pathogens (Masavuli et al., 2017).

“Molecular farming” is a term used to describe the utilization of plants or plant cells to produce recombinant proteins or other biologic drugs for use as cosmeceuticals, biopharmaceuticals, therapeutics, vaccines, and other reagents (Rybicki, 2020). Plants provide an enticing alternative system for the manufacture of VLP vaccines due to their potential to generate significant amounts of recombinant proteins at a minimal cost, their eukaryotic production machinery for the PTM and correct protein folding and assembly, and their low risk to introduce adventitious human pathogens. Plants do not demand the installation of costly fermentation facilities for the production of biomass, nor do they necessitate the establishment of duplicate facilities for scale-up production. Unlike microbial fermentation, plants have the ability to carry out N-glycosylation as a glycoprotein PTM (Chen and Lai, 2013). Engineered VLP-forming human or animal virus capsid proteins expressed in plant cells include human norovirus CP, HPV L1 protein, hepatitis B core and surface antigens, HIV Gag polyprotein, and HA protein of influenza virus (Rybicki, 2020).

Several studies have extensively discussed the concepts for constructing VLPs in various plant ESs, efficient growth of VLPs in plant hosts, and the self-assembly of multiple structural proteins of viruses in plants. Santi et al. (2008) produced properly assembled recombinant Norwalk virus VLPs in leaves of Nicotiana benthamiana utilizing a TMV-derived transient ES. Young et al. (2015) generated trackable hemagglutinin based VLPs that allowed them to examine particle assembly in plants and the interaction of VLPs inside the mammalian immunological system. van Zyl et al. (2016) investigated the production of bluetongue virus VLPs in N. benthamiana through Agrobacterium-mediated transient expression, which is an inexpensive system. Similarly, Veerapen et al. (2018) transiently expressed VLPs of the foot-and-mouth disease virus in N. benthamiana. Diamos and Mason (2018) produced norovirus VLPs in a plant-based system using modified geminiviral vectors. Recently, Rosales-Mendoza et al. (2020) presented a perspective in developing VLP vaccines based on plants against SARS-CoV-2, which is responsible for the COVID-19 pandemic.

Plants for the production of the VLP platform are not entirely acceptable due to comparatively lower production levels of VLP than mammalian ESs and plant-specific N-glycosylation of glycoproteins (Kim et al., 2014). Nonetheless, the recent creation of novel plant ESs, as well as advancements in plant glycoengineering, have both resolved these challenges (Chen and Lai, 2013). Recent advancements in plant glycoengineering permit human-like modification of glycol and optimization of desirable glycan structures to improve the functionality and safety of recombinant pharmaceutical glycoproteins (Kim et al., 2014).

Zika virus (ZIKV) is a small-enveloped mosquito-borne neurotropic positive-strand RNA virus of the family Flaviviridae (Lin et al., 2018; Shanmugam et al., 2019). ZIKV infections were associated with acute prenatal abnormalities like microcephaly in neonates born to infected mothers, and also Guillain-Barré syndrome (GBS) in adult people (Alvim et al., 2019). Currently, no cure or vaccine is commercially available for effective therapy or treatment, so vaccine development against ZIKV is of great importance (Boigard et al., 2017).

ZIKV has an RNA genome with only one open reading frame, and a solitary polyprotein is formed. This protein-strand is cut into 7 non-structural proteins (NS5, NS4B, NS4A, NS3, NS2B, NS2A, and NS1) and 3 structural proteins (C, prM, and E) by cellular and viral proteases. E-protein is engaged in the binding of viral particles as well as its fusion (Boigard et al., 2017). E-protein has three domains structure, like enveloped-domain-I also known as ED-I, then ED-II, and ED-III. Humoral response mainly targets the fusion-loop belonging to ED-II. Antibodies against fusion-loop epitopes enhance the uptake of DENV by Fcγ-receptors (Shanmugam et al., 2019). So, this protein may serve as a possible target for vaccine development (Shanmugam et al., 2019).

Many VLPs for ZIKV have been developed (Boigard et al., 2017). Garg et al. (2019) compared different VLPs-vaccine for ZIKV in mice and showed that CprME-VLPs (Capsid-preMembrane-Envelope) gave better results than prME-VLPs (preMembrane-Envelope). Cell lines could not be generated using CprME- because co-expression of protease NS2B-3 is needed. In order to get rid of this barrier, a bicistronic vector was generated that uses IRES-sequence to produce both NS2B-3 and CprME-VLPs. Alvim et al. (2019) demonstrated the continuous expression of ZIKV-VLPs by HEK293-cells. In short, the cell lines constitutively producing Zika-VLPs are ideal for developing a vaccine.

It’s a severe and periodic infectious disease caused by CHIKV (chikungunya-virus) transmitted by a carrier mosquito. Symptoms are high fever, skin rash, etc. No vaccine is available at present, but many VLP-vaccine candidates are under development in different stages (Zhang et al., 2019). Protection from multiple strains of this virus can be conferred by self-assembled VLPs produced as a result of selective expression of CHIKV envelope and capsid proteins (Kramer et al., 2013).

Arévalo et al. (2019) reported 100% protection in adult mice against CHIKV infection when unadjuvanted CHIKV-VLPs were used. Similarly, Metz et al. (2013) compared three different vaccines in mice that were produced in insects by recombinant baculoviruses produced sE1, sE2 CHIKV, and CHIKV (VLPs). One-half of E1 and E2 immunized mice survived to show incomplete protection when compared with VLP-immunized mice.

Akahata and Nabel (2012) uncovered that variable CHIKV structural proteins expression results in VLPs, which mimic replication-competent alphaviruses. This vaccine caused the induction of neutralizing antibodies in large amounts against multiple strains of this virus and conferring complete protection. VLPs were easily produced from CHIKV 37997-strains compared to CHIKV-OPY-1 strain- VLPs despite high amino-acid-sequence similarity. Knowledge of mechanisms involved in CHIKV VLP production will help in other vaccine development also enhancing their range of application for other pathogens. Furthermore, VLP-production was affected by amino acid 234 of E2 in an acid-sensitive region. Mutations in the acid-sensitive region and pH changes also enhance the yield of VLPs.

Influenza virus (Iv) infections are the leading cause of chronic human respiratory symptoms, leading to severe public health outcomes about endemic and seasonal infections and even result in unpredictable pandemic outbreaks (Lai et al., 2019). Iv is an enveloped, segmented, negative-sense RNA virus, which belongs to the family Orthomyxoviridae. To date, four types of Ivs have been identified, classified according to the presence of their core proteins: A, B, C, and D. Three Iv types, A, B, and C are pathogenic to human cells and cause severe infections, with Iv type A and B being the most prevalent circulating types. The main surface glycoproteins of influenza viruses are neuraminidase (NA) and hemagglutinin (HA) (To and Torres, 2019).

HA, the key antigen involved in infection, attaches to the residues of sialic acid on the cell membrane surface, facilitating the Iv to enter the host cell. NA appears to be less frequent on the viral surface as compared to HA, with a generally observed NA:HA ratio of 1:4. Its enzymatic function is critical in the cleavage of sialic acid, thereby facilitating viral release from the infected host cell surface. NA activation also enables the successful penetration by influenza to mucus through a mechanism involving cleavage of sialic (Buffin et al., 2019). Kim et al. (2019) investigated the cross-protective efficiency and immunogenicity of VLP containing NA (N1 VLP) derived from the 2009 H1N1 influenza viral pandemic and compared it to inactivated split influenza vaccine. Mouse immunized with the N1 VLPs was able to induce virus-specific Ab responses as well as cross-reactive NA inhibition activity, while strain-specific hemagglutination inhibition test was induced by inactivated split vaccination. Mice vaccinated with N1 VLPs led to the development of cross-protective immunity to antigenically various Ivs, as measured by changes in bodyweight, pulmonary viral titers, infiltration of innate leukocytes, cytokines and Ab secretory cells, and germinal center B-cells. Furthermore, in naïve mice, the immune sera of N1 VLPs conferred cross-protection. Immunity induced by N1 VLPs was neither impaired nor diminished in mice lacking the Fc receptor γ-chain. These findings indicate that NA representing VLPs, along with the current vaccination of influenza, may be further improved and exploited as an important candidate for cross-protective vaccines.

Recently, Kirsteina et al. (2020) studied and examined the efficacy and immunogenicity of an array of widely protective prototypes of Iv vaccine focused on both influenza triple matrix protein 2 ion channel (3M2e) and tri-stalk antigens incorporated into phage AP205 VLPs. VLPs that contained the 3M2e antigen alone stimulated protection in mice toward both standard homologous as well as heterologous virus challenge. The combination of both conserved antigens of the influenza virus into an individual VLP resulted in complete protection against a high dose of homologous influenza H1N1 infection in mice.

Human papillomaviruses (HPVs) are members of the Papillomaviridae family of tiny, circular, double-stranded DNA viruses that cause cervical cancer, the world’s second most fatal disease in women after breast cancer (Uddin et al., 2019; Burley et al., 2020). Currently, two VLP-based vaccines are commercially available for inhibiting warts and treating cervical cancers caused by HPV. These include Cervarix by GlaxoSmithKline (GSK) Pharmaceuticals and Gardasil by Merck Pharmaceuticals. Both vaccines consist of the immunogenic major CP (L1) VLPs of HPV 16 and 18, with Gardasil also having 6 and 11 VLPs (Uddin et al., 2019).

The drawback of using L1 protein as an antigen for the VLP vaccine is that it is not conserved among various HPV serotypes. Conversely, the minor CP (L2) is highly conserved across all HPV serotypes and has long been considered a major potential target antigen for developing an HPV vaccination with broad protection. In contrast to CP (L1), the CP (L2) cannot be used for VLP production and is, therefore, less immunogenic (Yadav et al., 2020). The reasons for the low immunogenicity of L2 proteins are due to their slight representation relative to L1, as well as the fact that L2 is mainly buried beneath the surface of the capsid, where it contacts the surface of the cell in vitro or the basement membrane in vivo and induces a conformational shift (Huber et al., 2017).

Several methods have been applied to improve and enhance the L2 peptides low immunogenicities, such as concatemeric fusion peptides consisting of L2 epitopes of various types of HPV, combined with immune activating TLR agonists or using repeated surface arrays of epitopes of L2 on particulate immunostimulatory platforms, such as VLP of TMV, bacteriophages MS2 or PP7, Adeno-associated virus, Lactobacillus casei, or bacterial thioredoxin (Huber et al., 2017).

Another promising strategy to improve L2 low immunogenicity is the presentation of L2 epitopes on the surface via VLPs assembled from HPV L1-L2 chimeric proteins. It was achieved through the genetic insertion of highly conserved B cell epitope RG1 of HPV16 L2 into the surface loop (DE) of the protein HPV16 L1 leading to its multivalent (360x) immunogenic display on the surface of VLP, whereas the conformational neutralization antigenic determinants of the HPV16 L1 scaffold remained mostly preserved. Immunizations with this chimeric RG1-VLP caused high type-specific HPV16 titers and extensive cross-neutralization of heterologous mucosal and distantly associated cutaneous HPVs (Schellenbacher et al., 2009, 2013).

Pouyanfard et al. (2018) described the thermostable thioredoxin vaccine development based on a single-peptide capable of carrying L2 polytopes from up to 11 various types of HPV. The antigens of the L2 polytope exhibit exceptional capabilities regarding the robustness and protection of the elicited immunological responses. To further boost and enhance immunogenicity, the polytope antigen L2 of thioredoxin was fused with a heptamerization domain. Protective responses to all 14 oncogenic types of HPV, as well as the lower risk HPV types (6 and 11), and a range of cutaneous HPVs were achieved in the final design of the vaccine.