With the ability to integrate biological data and computational analysis, synthetic biology has significantly contributed to move vaccine development beyond the constraints of Pasteur, assisting in the design of novel biological systems with enhanced efficacy and safety as well as reducing vaccine production times (Ruder, Lu, & Collins, 2011) . With today's focus gearing towards using VLPs, not only as vaccines of unmodified viral assemblies against parental viruses but also as scaffolds for display of heterologous antigens Roldão et al., 2010) , synthetic biology has become a centerpiece in VLP vaccine engineering. Seconded by a multitude of tools, such as omics technologies, structural biology, system immunology, and bioinformatics and computational biology, one can now screen for pathogen-specific antigens with high immunogenic potential and apply that information to rationally design modern VLP vaccines ( Figure 1 ).
Influenza is one of the best examples on how omics, more precisely genomics, is revolutionizing vaccine design. Dedicated databases detailing complete and accurate influenza genomic information have been created and can be readily accessed worldwide (McHardy & Adams, 2009 ). In the last decade, this vast wealth of data has been translated into knowledge, which, in conjunction with synthetic biology, has aided the design of VLP vaccine candidates (Prabakaran et al., 2010; Pushko et al., 2011; Pushko et al., 2017) . These novel biological entities are undoubtedly safer with the potential to be more broadly reactive than their previous counterparts (i.e., traditional, commercially available live attenuated influenza vaccines).
Several genomic applications aid in the identification of novel antigens (Liao et al., 2017) , namely, reverse vaccinology and comparative and functional genomics. Reverse vaccinology combines genomics with proteomics and bioinformatics to identify virtually all potentially protective antigens from coding regions within the genome (Bambini & Rappuoli, 2009; Rappuoli, 2007) . Comparative genomics is primarily used to design broadly protective vaccines as it allows comparison of conserved and variable open reading frames within the same species. Functional genomics allows the identification of protein function based on reverse genetic evaluation (mutations and knockouts) or gene expression analysis (transcriptomics; Bagnoli et al., 2011; Bambini & Rappuoli, 2009; Rappuoli, 2007; Sette & Rappuoli, 2010) . Although genomics per se has contributed significantly to the design of many vaccine candidates, such as influenza (Prabakaran et al., 2010; Pushko et al., 2011; Pushko et al., 2017) , malaria (Draper et al., 2015; Y. Wu, Narum, Fleury, Jennings, & Yadava, 2015) , and human immunodeficiency virus (HIV; Calazans, Boggiano, & Lindsay, 2017) , its full potential for vaccine development can only be realized when integrated with proteomics and immunomics (Bagnoli et al., 2011) . Moreover, the coordinated deployment of these omics technologies along with miniaturized bioprocess screening in, for example, microbioreactor and microfluidic cell culture systems can aid developability assessment leading to accelerated manufacture (Bhambure, Kumar, & Rathore, 2011; Gong & Lei, 2014; Hemmerich, Noack, Wiechert, & Oldiges, 2018) .
Immunomics specifically addresses the interface between the host immune system and the pathogen proteome. It studies the subset of pathogen-derived proteins or epitopes that are recognized by the host immune system. This information can be used to validate antigens identified from in silico and/or in vitro approaches by evaluating whether they are targets of clinically relevant immune responses (i.e., they stimulate the production of specific cytokines or activate specific cell types; Kuleš et al., 2016) . An example of immunomics application to VLP vaccine design is GlaxoSmithKline's RTS,S vaccine, the most advanced malaria vaccine candidate. RTS,S is a chimeric VLP in which a large portion of the C-terminal of Plasmodium falciparum-derived circumsporozoite protein (CSP) is displayed on a protein scaffold (hepatitis B surface antigen; Y. Wu et al., 2015) . In combination with systems biology (genomics and proteomics), immunomics was used to highlight regions within CSP that were immunogenic and protective in humans and, thus, indispensable to incorporate into a chimeric VLP. From the many candidates identified, only one proved to be successful in Phase IIa/b efficacy trials (Draper et al., 2015; Kazmin et al., 2017) .
Structural information can be used to reconfigure and engineer conserved epitopes to expose areas that exhibit high immunogenicity or to insert multiple immunodominant epitopes within the same VLP platform (Liljeroos, Malito, Ferlenghi, & Bottomley, 2015) . These strategies can broaden the immune response or enhance the existing response to weak immunogenic antigens. Identifying conformational epitopes and studying their interactions with the immune system can provide significant information for rational antigen design (Anggraeni et al., 2013; Mulder et al., 2012) .
Encouraged by recent results from HIV-1 VLPs (C. Zhao, Ao, & Yao, 2016) , structural biology is emerging as a powerful tool to assist in the rational design of a modern HIV VLP vaccine. Novel protective epitopes can now be identified in conformational epitope mapping studies via structural biology (Liljeroos et al., 2015; Malito, Carfi, & Bottomley, 2015) . In addition, broad and potent HIV antibodies discovered in the pool of antigen-specific memory B cells using structural biology, highlight novel sites of vulnerability on HIV envelope glycoprotein epitope (J. Huang et al., 2014) . When incorporated into HIV-1 VLPs, these antigens can be strategically modified to insert the extended 2F5, 4E10 epitope and membrane proximal external region (MPER) of HIV-1 gp41 (Zhai, Zhong, Zariffard, Spear, & Qiao, 2013) providing a better display of the conserved CD4 binding site and capturing broadly neutralizing antibodies (Ingale et al., 2014) .
Structural biology is equally informative for VLP engineering as the capsid proteins forming each VLP need to be correctly folded to (a) (b) F I G U R E 1 Design tools for VLP vaccine engineering. Multitude of tools and recent advances in synthetic biology enable screening for pathogenspecific antigens with high immunogenic potential and engineering of VLP function. (1) Omics technologies enable rapid identification and discovery of novel/potential vaccine antigens. (2) Structural biology and (3) system immunology assist rational reconfiguration and engineering of epitopes/VLPs for enhanced immunogenicity. (4) Bioinformatics and computational biology accelerate data analysis and translation into applicable knowledge. (a) Engineering VLP function on different types of VLP. While nonenveloped VLPs are commonly engineered using genetic engineering or chemical conjugation, enveloped VLPs rely on pseudotyping for function engineering. (b) VLPs can be engineered to offer broader immunogenicity, improved immunogenicity, or enhanced stability. Broadly immunogenic VLPs can be obtained by displaying multiple antigenically distinct epitopes (Pushko et al., 2011; Schwartzman et al., 2015) , highly conserved epitopes (Krammer, 2015; Wiersma et al., 2015) , or computationally optimized epitopes (Carter et al., 2016) within a single VLP. Improving VLP immunogenicity can be achieved by incorporating immunomodulatory agents, such as dendritic cells targeting antibodies into particles structure (Rosenthal et al., 2014) . VLP stability can be enhanced by modulating particles formulation (Collins et al., 2017; Lua et al., 2014 
Systems immunology is an emerging area of research that uses a broad and integrated, multilevel approach to study the immune system to identify immune correlates of protection or immunogenicity signatures (Davis, Tato, & Furman, 2017) . Combined with recent technological advances in human immunology, systems immunology can provide guidance for rational vaccine design. Systems immunology allows the assessment of most cell types of the immune system (including specialized B and T white blood cells), their state, function, signaling molecules, and encoding genes (Bird, 2017) . This accumulation of data captures a snapshot of the human immune system, providing valuable information for the creation of human immune response models that may later be translated into improved vaccine design (Davis et al., 2017) . A systems immunology approach was previously undertaken to investigate the immune response to the live attenuated yellow fever vaccine YF-17D (Hou et al., 2017; Muyanja et al., 2014) . Following immunization, comprehensive characterization of the immune system was performed providing insights into the vaccine's mechanism of action. A similar method was later applied to the malaria vaccine RTS,S, where a systems-level approach led to the identification of molecular and cellular signatures associated with protection and immunogenicity unique to this VLP-based vaccine (Kazmin et al., 2017) . Systems immunology may also play an important role in designing vaccines capable of stimulating parts of the immune system not addressed by current vaccines (Davis et al., 2017) . VLPs are ideal testing candidates for systems immunology approach due to their ability to stimulate Bcell-mediated immune responses, as well as CD4 proliferative responses and cytotoxic T lymphocyte responses (Gause et al., 2017) .
The correct identification of epitopes that stimulate an immune response is crucial for the design of novel immunogens. These epitopes are regions within the antigen that are recognized by B-and T-cell receptors (Patronov & Doytchinova, 2013) . Potential B-and Tcell epitopes can be mapped using bioinformatics and computational biology tools, however, without recent improvements in immunological characterization methods, the identification of epitopes that optimally stimulate the human immune response becomes challen- 
Bioinformatics and computational biology tools have the potential to accelerate data analysis and translate results into applicable knowledge, fostering the discovery of new lead antigens by reducing the number of empirical experiments (He & Xiang, 2013) . Vaccine design is an inherently complex and laborious process but software, algorithms, and databases outlined below have the potential to streamline vaccine development via identification of candidate antigens that may otherwise have been overlooked.
Epitope mapping is essential for designing vaccines capable of mounting a robust T-and B-cell response. 
Bioinformatics and computational biology can also assist in the discovery of conserved epitopes through sequence variability analysis. in/raghava/antigendb/) stores sequences, structures, origins, and epitopes of pathogen antigens (Ansari, Flower, & Raghava, 2010) .
The Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was recently developed to overcome the challenges associated with antigenic diversity in influenza subtypes (Carter et al., 2016) . This in silico approach, which uses consensus building to generate a number of antigen candidates termed COBRA antigens, was used to identify HA antigens that were broadly protective against VLPs are divided into two main groups, enveloped and nonenveloped. Enveloped VLPs are self-assembling capsids, which acquire a lipid layer when budding from their host cells. This layer is absent in nonenveloped VLPs (Mateu, 2011) . Insertion of heterologous antigens (otherwise known as modularization) into nonenveloped VLPs is mainly achieved through genetic fusion or chemical conjugation (Peacey, Wilson, Baird, & Ward, 2007) . The size of the insert has implications for VLP assembly and correct presentation of the antigen. Small peptide epitopes are easily inserted into VLP structures without affecting VLP assembly, which can often occur when modularizing whole or large protein domains. Genetic fusion is the most popular method despite being time-consuming and error-prone (Mateu, 2011) . Chemical conjugation supports the insertion of large antigens in preformed
VLPs and these modifications are performed on naturally occurring conjugation sites using chemical crosslinkers or enzymes (Patel & Swartz, 2011; Tang, Xuan, Ye, Huang, & Qian, 2016) . The downside of chemical conjugation is the incurred cost as it requires the production of both the VLP and the epitope(s) as well as conducting the chemical conjugation (Chackerian, 2007; Smith, Hawes, & Bundy, 2013) . Recently developed technologies have started to address the cost and technological challenges. An example is the "Plug and Display" system, a technology based on two proteins, "the tag" and "the catcher," which react irreversibly when in close proximity of each other. When "the catcher" is fused to a VLP and "the Tag" is fused with a vaccine target, these form a two-component VLP vaccine ready to use (Brune et al., 2016) . Enveloped VLPs can present heterologous membrane proteins (i.e., glycoproteins) in their native configuration on top of a selfassembling capsid protein(s) without the need to engineer both epitope and capsid structures in a process called pseudotyping (Chua et al., 2013; Kirchmeier et al., 2014) . With pseudotyping, one can alter the VLP stability or even its tropism (Cronin, Zhang, & Reiser, 2005 ; K. Palomares et al., 2013) . In addition, transmembrane domains within foreign viral envelope proteins can be replaced with transmembrane domains of specific viruses (e.g., vesicular stomatitis virus) to improve pseudotyping efficiency and immunogenicity (Kirchmeier et al., 2014) . Enveloped VLPs, such as retro-and lenti-VLPs, have shown promise as vaccines candidates against diseases, such as Influenza, Malaria, or Dengue virus (Chua et al., 2013; Pitoiset, Vazquez, & Bellier, 2015) .
The engineering of VLP has long been a complex process and often unsuccessful, as the insertion of small peptides can disrupt the VLP structure. The field has evolved and now VLP chimeras are used in both fundamental and applied research (Mateu, 2011; Murata et al., 2009) . A successful example of the insertion of large peptides is the VLP derived from flock house virus that was engineered to carry a receptor domain and could be used as an anthrax antitoxin as well as a vaccine (Manayani et al., 2007) .
Circulating viruses that exhibit high antigenic variability and high mutation rates, such as influenza, pose substantial challenges for current vaccination strategies . High immunogenicity is a desirable vaccine attribute that potentially translates to protectivity against target infections. To enhance immunogenicity, modularized antigens must be strategically inserted to maximize their presentation to the immune system.
has been previously reported (De Filette et al., 2008; Haynes et al., 1986 ). However, low surface expression or the inability of peptides to adopt native conformation can result in weak immunogenicity at these insertion sites (Schödel et al., 1992) . Platforms based upon HBcAg, papillomavirus (bovine and human), Flock House virus (FHV), and others, are thus engineered to take advantage of surfaceexposed loops that demonstrate enhanced immunogenicity (Murata et al., 2009; Slupetzky et al., 2007; Ye et al., 2014) . Peptides inserted into exposed loops protrude from the surface of VLPs making them more accessible to the immune system. Short peptides displaying relatively simple structures pose little problem for modularization within exposed loops, yet those with more complex structures require further platform engineering (Anggraeni et al., 2013) .
Incorporation of epitope scaffolds into exposed loops of VLPs Display of whole protein domains on the surface of VLPs allows presentation of multiple antigenic epitopes and increases the likelihood that epitopes will adopt their native conformation. Their large size, however, can cause steric hindrance resulting in compromised VLP assembly (Lua, Fan, Chang, Connors, & Middelberg, 2015) . As such, several strategies have been developed to specifically modularize large antigens. Long, flexible glycine-rich linkers were engineered to flank the antigen in the c/e1 loop of the HBcAg platform, allowing spatial separation and enabling proteins of up to 238aa to be inserted (Kratz, Bottcher, & Nassal, 1999) . Though effective, optimal linker length for different antigens needs to be empirically determined. Steric hindrance can also be addressed in some cases by reducing the antigen content on the surface of the VLP. For example, Peyret et al. (2015) engineered a single polypeptide composed of an unmodified HBcAg fused to a chimeric HBcAg. A dual expression construct was also devised to coexpress unmodified murine polyomavirus (MuPyV) VP1, with antigen-modularized VP1 displaying an 18 kDa rotavirus antigen Tekewe, Fan, Tan, Middelberg, & Lua, 2017) .
Although such methods can lead to successful VLP assembly, it is possible that increasing the antigen mass, whereas reducing the antigen number may be reflected in lower immunogenicity, as is seen CHARLTON HUME ET AL.
with the synthetically derived RTS,S malaria vaccine (Pitoiset et al., 2015) . Nevertheless, this trade-off is inevitable as the mass of the antigen increases relative to the carrier, necessitating an understanding of the optimization domain.
Display of large antigens has been reported using novel platforms based upon the HPV 16 L1 protein and the Acinetobacter phage, AP205 (Thrane et al., 2015; Thrane et al., 2016; Brune et al., 2016) . and MuPyV VP1 proteins has led to the development of capsomere platforms (Middelberg et al., 2011; Schadlich et al., 2009) . Removal of the carboxyl termini from each protein yields capsomeres incapable of forming VLPs in vivo. MuPyV capsomeres display increased stability, although less immunogenic than VLPs when administered with adjuvant they are just as effective (Middelberg et al., 2011) .
Furthermore, a quantitative process study reported that when produced in E. coli, the MuPyV capsomere platform is capable of producing 320 million vaccine doses in 2.3 days at low cost highlighting its suitability as a rapid response and low-cost vaccine platform .
The safe and robust influenza M1 platform further illustrates the aforementioned, broadly immunoprotective capabilities of VLP platforms. Multiple subtypes of HA antigens have been displayed on M1-VLPs either individually (Schwartzman et al., 2015) or simultaneously (Pushko et al., 2011; Sequeira et al., 2017; Tretyakova, Pearce, Florese, Tumpey, & Pushko, 2013) . M1-VLP based vaccines afford protection against virus challenge in multiple species (Liu, Massare, et al., 2015; Pyo et al., 2012) (2011) F I G U R E 2 Application of synthetic biology to VLP vaccine platforms.
(1) Enhanced immunogenicity of peptides is achieved through their insertion into exposed loops of viral capsid proteins (Murata et al., 2009; Slupetzky et al., 2007; Ye et al., 2014) . (2) The structural properties of complex peptides are maintained through the incorporation of epitope scaffolds into exposed loops (Schneemann et al., 2012) . (3) Large antigens are modularized onto VLP vaccine platforms using long flexible linkers to maintain structural separation between the viral capsid protein and the antigen (Kratz et al., 1999) ; or onto preformed VLPs using plug and play technologies, such as SpyCatcher/SpyTag (Brune et al., 2016) and AviTag (Thrane et al., 2015) . (4) Dual expression of modified and unmodified viral capsid proteins reduces steric hindrance and permits VLP assembly (Tekewe et al., 2017) . (5) The SplitCore system permits modularization of antigens with an extended structure through the coexpression of modified and unmodified HBcAg core fragments (Walker, Skamel, and Nassal, 2011) . (a) Synthetic production of capsomeres minimizes host cell contaminants reducing required bioprocessing steps (Chuan et al., 2010) . and a stable cell line can be adopted. Such a strategy has proven already to be successful for the production of multivalent influenza VLPs using an insect High Five cell-based platform (Sequeira et al., 2017) .
Vaccine production scale, cost, and purity vary depending on VLP purification processes. Centrifugation, depth, and tangential flow filtration, used for initial clarification and concentration steps, are easily scaled. At the industrial level, chromatographic methods are used to remove host cell and DNA impurities as density gradients and ultracentrifugation are not easily scalable. Anion exchange and SEC are time-consuming and costly, highlighting the need for more efficient and cost-effective methods. Nonchromatographic strategies based upon aqueous two-phase systems (ATPS), a process presently used for enzyme production at the industrial level, are being developed for VLP purification. High yields of rotavirus VLPs have been purified from insect cell supernatant using ATPS, although purity was relatively poor (Benavides et al., 2006) . More recently, single and multistep ATPS were used to purify human B19 parvovirus-like particles from insect cell lysates (Effio et al., 2015) .
Purity levels were greater than 90%, yet this appeared to be at the expense of yield. Monolith technology presents a rapid and scalable method that offers distinct advantages over the classical packed-bed chromatography (Vicente et al., 2011) . HBsAg VLPs from yeast homogenate was effectively purified using a hydroxyl derivatized monolith (Burden, Jin, Podgornik, & Bracewell, 2012) and when compared to density gradient centrifugation, an anion-exchange monolith yielded 220-fold more HIV-1 gag VLPs from Chinese hamster ovary (CHO) cell supernatant (Steppert et al., 2016) .
Sulfated cellulose membrane absorbers offer significant improvements over conventional ion exchangers membrane absorbers (Carvalho, Fortuna, et al., 2017) . As they are easily scaled and reduce the number of required processing steps they may qualify as a generic purification platform for VLP-based vaccines. These technol- (Carvalho, Moleirinho, et al., 2017 VLP vaccine platforms provide opportunities to overcome several limitations of traditional vaccine manufacturing. Substantial variation between antigen characteristics of traditional vaccines and their infectious nature adds significantly to production cost as they demand specialized production facilities and tailored manufacturing processes (S. Plotkin, Robinson, Cunningham, Iqbal, & Larsen, 2017) . The standardized production of noninfectious, generic vaccine platforms (used to display various antigens) would reduce process variation (i.e., platform remains constant) and provide grounds to establish multiproduct facilities. Streamlining of upstream and downstream processes is likely to increase efficiency, reduce laboratory waste, and permit cost savings through bulk buying of equipment and reagents (Konstantinov & Cooney, 2015) . Predictable bioprocesses may allow the standardization of facility infrastructure and the use of single-use technologies, already in use for some VLP vaccines (Roldão et al., 2010) . Disposable technologies offer several advantages over traditional stainless steel equipment including lower initial investment and operating costs, elimination of cross-contamination, and flexibility in terms of scale (Lopes, 2015; Shukla & Gottschalk, 2013) . Combining disposable technologies with VLP platforms offer advantages on multiple levels and would facilitate VLP translation into the industry.
The increased cost and regulatory uncertainty associated with producing new vaccines have long been challenges for vaccine manufacturers (Ulmer, Valley, & Rappuoli, 2006) . The intricacies of vaccine manufacturing demand a substantial amount of expertize and contribute significantly to cost (S. Plotkin et al., 2017) . Expenses associated with vaccine R&D, testing and manufacturing as well as a poor profit margin compared with current drug markets, have led to a dramatic fall in funding from profit-driven pharmaceutical companies (Offit, 2005) . However, this decline is thought to be premature. As demonstrated throughout this review, VLP technologies are advancing rapidly with a strong focus on developing platform capabilities and improving VLP production yields to enable rapid production of safer and cheaper vaccines, particularly when combined with single-use bioprocessing systems. This progress is set to continue with financial investment provided from "not-for-profit," sources, such as national governments, international organizations, and philanthropic bodies. In particular, Gavi, the vaccine Alliance (Balaji, 2004) and the Bill & Melinda Gates Foundation (www.gatesfoundation.org) are motivated to deliver affordable vaccines to third world countries that are the world's biggest vaccine market. To ensure vaccine purity, safety, efficacy, and stability, regulatory authorities govern every stage of vaccine manufacturing from raw materials and production processes to clinical trials and beyond. This includes the assessment and certification of safe and viable manufacturing processes (S. Plotkin et al., 2017) . Meeting regulatory requirements can be a complicated process, particularly when manufacturing vaccines for overseas markets or utilizing newly developed bioprocesses. By streamlining upstream and downstream processes and utilizing a generic VLP base, VLP platform technology is expected to reduce the regulatory load of individual vaccines given that regulations for the base and its purification will become well characterized. VLP platforms may even fast track vaccine delivery in response to pandemic circumstances.
Vaccinology is experiencing an impressive technological revolution, enabling to move vaccines development beyond the rules of Pasteur (empirical approach), using data-rich disciplines, such as systems biology, immunology, or computational biology to assist rational vaccines design (modern approach). Indeed, the last decade has witnessed a trend toward the use of alternative vaccine designs to attenuated pathogens, having VLPs emerged as a powerful and versatile platform for their production. Today, VLPs are being used not only as vaccines of unmodified viral assemblies against parental viruses but also as scaffolds for displaying heterologous antigens. In addition, tremendous investments have been made to develop new technologies capable of (a) deciphering pathogen biology and vaccine mechanistic responses, and (b) storing and curating extracted data into biological references databases. Machine learning algorithms can then use this information for epitope prediction and structure-based modular design. Complemented with synthetic biology, this information will provide the basis for (a) engineering VLP functions and (a) developing generic VLP platforms offering not only the potential for streamlined bioprocesses, parallel infrastructure, and predictable biosafety but also the ability to manufacture vaccines against unrelated viruses or pathogens from other sources (i.e., bacteria and parasitic protozoans). Although unable to deliver any marketable product to date, these technologies have massive potential to provide, in near future, solutions against untargeted infectious agents (e.g., antibiotic-resistant bacteria, HIV, malaria, or tuberculosis), and most importantly to reduce vaccine development times and manufacturing cost associated with current vaccine platforms.
The authors acknowledge the support from Australian Research 