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ScienceDirect Pathogen-like particles: biomimetic vaccine carriers engineered at the nanoscale Joseph A Rosenthal1, Linxiao Chen2, Jenny L Baker2, David Putnam1,2 and Matthew P DeLisa1,2 Vaccine adjuvants are an essential component of vaccine design, helping to generate immunity to pathogen antigens in the absence of infection. Recent advances in nanoscale engineering have created a new class of particulate bionanotechnology that uses biomimicry to better integrate adjuvant and antigen. These pathogen-like particles, or PLPs, can come from a variety of sources, ranging from fully synthetic platforms to biologically derived, self-assembling systems. By employing molecularly engineered targeting and stimulation of key immune cells, recent studies utilizing PLPs as vaccine delivery platforms have shown great promise against highimpact, unsolved vaccine targets ranging from bacterial and viral pathogens to cancer and addiction. Addresses 1 Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA 2 School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA Corresponding authors: Putnam, David ([email protected]) and DeLisa, Matthew P. ([email protected])

Current Opinion in Biotechnology 2014, 28:51–58 This review comes from a themed issue on Nanobiotechnology Edited by Jonathan S Dordick and Kelvin H Lee

0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.11.005

Introduction Vaccines have a tremendous global impact on human health, from the near-eradication of several important diseases to the annual prevention of recurrent epidemics in a variety of vulnerable populations. However, most vaccines in use today were developed using techniques pioneered more than 100 years ago and do not reflect the full potential of the field. With the introduction of emerging biotechnologies such as genetic engineering, rapid advances have been made in the design, synthesis, and application of modern vaccines against a myriad of preventable diseases. A modern vaccine has two simple goals: replace the vaccine target with a simplified molecular identity (antigen) that the immune system can use to recognize the target in the future and replace the inherent immunostimulation of a pathogen’s infection with a less dangerous stimulus (adjuvant) [1–3,4]. www.sciencedirect.com

However, accumulating evidence indicates that the way in which the adjuvant mediates the interface between the antigen and the immune system is substantially more complex than previously believed [5–8,9]. This becomes especially important if the antigen is no longer an intact pathogen but instead is an individual subunit derived from the pathogen (e.g. a recombinant protein), as is increasingly the case for new vaccines. To generate a successful immune response, a pathogen or pathogenic subunit needs to have meaningful interactions with three groups of inter-operating cells (Figure 1): innate immune cells, professional antigen presenting cells (APCs), and adaptive effector/memory cells [10–15]. These interactions, primarily mediated by immunological synapses and cytokine intermediaries, are non-trivial in their complexity and interconnectivity. A successful adjuvant needs to be capable of productively stimulating this network while at the same time avoiding lasting damage to the host. Recently, nanotechnology has been applied to address the clear biomimicry challenge inherent to the next generation of more potent and sophisticated adjuvants. Although early adjuvants, such as aluminum hydroxide, have been successful in stimulating the immune response, the advent of nanoscale antigen delivery platforms has catalyzed the transition from these simple immunoactive chemical agents to adjuvants that can engage and direct immune responses more akin to the employed by pathogens themselves methods [1,6,9,12,14–17]. In this review, we will focus on a particular classification of nanoscale antigen carriers, namely pathogen-like particles, or PLPs (Figure 2). PLPs serve a simple purpose with a complex execution: to present antigen and adjuvant together in a manner that synthetically mimics the pathogen-immune cell interaction. Three general classes of PLPs have emerged over recent years — synthetic particulate systems, virus-like particles, and bacterial outer membrane vesicles — each of which illustrates the power of nanoscale engineering applied to vaccine delivery.

Synthetic particulate vaccines Soluble antigens, independent of some larger delivery vector, suffer from reduced uptake by APCs and poor immunogenicity. These fundamental flaws have led to the development of particle-based vaccine carriers that can more closely mimic the physiochemical characteristics Current Opinion in Biotechnology 2014, 28:51–58

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Figure 1

Innate immunity cells

Activation and modulation

Recruitment and stimulation

Pathogen-like particle (PLP)

Adaptive effector/memory cells

Antigen presenting cells

Education and maturation Current Opinion in Biotechnology

Immunostimulation routes of pathogen-like particles (PLPs). PLPs have the capacity to co-deliver antigen and immunostimulation directly to innate immunity cells, antigen presenting cells (APCs), and adaptive cells. This facilitates the complete engagement of an effective immune response. Innate immune cells, such as granulocytes, are activated at the site of vaccine administration to recruit APCs, such as dendritic cells (DCs), due to the presence of molecular immunostimulators on the PLP. DCs can then migrate to secondary lymphoid tissue, where they can become stimulated directly by PLPs that have passively drained or been targeted to this site. In the tissue, naı¨ve adaptive cells, such as T-cells, are selected for either via presented antigen from activated APCs or by direct antigen delivery via PLPs. This leads to effector subtypes that mediate active immunity via cytokine secretion, antibody production, and cytotoxic delivery, as well as memory subtypes that preserve the immune response.

of natural pathogens, enhancing antigen delivery to the immune system [18]. PLP carriers can be formulated using nanoparticles created from biocompatible polymers, such as polylactic acid (PLA) and poly(lactic-co-glycolic) acid (PLGA), liposomes, and even simple lipid emulsions [19– 21]. Antigens are then either encapsulated by, or integrated onto the surface of, these particles. Particle-based delivery is a promising technology due to its ability to target APCs such as dendritic cells (DCs) and stimulate antigen uptake, simultaneously deliver antigens and co-stimulatory signals, and generate strong and distinct immune responses [22]. Furthermore, each of these characteristics may be finely tuned to the specific needs of the vaccine by varying the physiochemical attributes of the particle. Size and surface characteristics are two important considerations for targeting and stimulating antigen uptake by professional APCs such as DCs. For example, distinct populations of APCs may be targeted simply by modulating particle size. Smaller particles (20–200 nm) are able to more easily target DCs residing in lymph nodes by exploiting interstitial circulation to access the lymphatic Current Opinion in Biotechnology 2014, 28:51–58

system, whereas larger particles (0.5–2 mm) remain at the injection site and are taken up by peripheral DCs and tissue-resident macrophages [23,24]. Moreover, there is a strong relationship between the mode of particle uptake and the size of the particles themselves. Particles with small diameters (500 nm) are traditionally taken up by phagocytosis or macropinocytosis [25]. However, the correlation between immune response and particle size is more ambiguous for nanoparticle vaccines, and the literature currently lacks a well-defined optimal size range for such delivery vectors [26]. These discrepancies may result from other factors such as differences in materials used for the production of the particle. Efficient uptake can also be achieved by creating particles using materials that more closely mimic the properties of pathogen surfaces [27]. Liposomes and nanoscale lipid emulsions formed from cationic lipids (such as Novartis’ MF591 and GlaxoSmithKline’s AS031) have been shown to enhance immunity; the positively-charged liposomes more easily interact with the negatively charged cell surface of DCs. Similar results have been observed by constructing nanoparticles using cationic polymers [28– 30]. More sophisticated approaches towards DC-specific targeting include the conjugation of antibodies specific for certain externalized membrane targets, such as the endocytic receptor DEC205, to the surface of nanoparticles [31]. Maturation of vaccine-stimulated DCs, necessary for a successful immune response, requires not only antigen presentation but also the presence of co-stimulatory molecules. These molecules bind to pattern recognition receptors (such as Toll-like receptors, or TLRs), triggering DC activation and maturation when delivered in conjunction with the appropriate antigen [32]. Particlebased vaccines can therefore be modified to include such components in order to mimic stimulatory signals present on pathogens. Inclusion of these co-stimulatory molecules leads to activation of both cellular and humoral immune responses. TLRs are the most well-studied class of co-stimulatory receptors, and numerous synthetic TLR ligands have been identified and incorporated into nanoparticle vaccines. For example, CpG oligodeoxynucleotides (CpG-ODNs), which engage TLR-9, were shown to protect against West Nile encephalitis when co-delivered in a nanoparticle vector [20]. Similarly, polyI:C, a synthetic analogue of double stranded RNA, has been incorporated into nanoparticle-based vaccines as a TLR-3 agonist, mimicking viral pathogens [33]. Lipid A, an endotoxic component of Gram-negative bacteria, has been incorporated into liposomes and serves as an strong adjuvant and TLR-4 binder [21]. Monophosphoryl lipid A, the detoxified version of lipid A and an FDAapproved adjuvant, has the same immunomodulatory properties of lipid A while lacking the associated toxicity www.sciencedirect.com

Pathogen-like particle vaccines Rosenthal et al. 53

Figure 2

immunostimulators

Size (50-500 nm) antigens

Synthetic Particulate Systems Pro: highly tunable Con: limited complexity

Virus-like Particles Pro: simple biomimicry Con: limited engineerability

Outer Membrane Vesicles Pro: self-adjuvancy and recombinant engineerability Con: endotoxic content Current Opinion in Biotechnology

PLPs as tunable, nanoscale delivery platforms for vaccines. Three key characteristics drive engineerable PLP vaccine delivery: antigen, immunostimulators, and size. Antigens may be encapsulated and, therefore, deliverable intracellularly for subsequent presentation on major histocompatibility complexes, or surface displayed to better mimic interactions between a pathogen and an immune cell. Additionally, the display of immunoactive surface ligands, such as TLR agonists or cofactor antibodies, or the incorporation of soluble factors, such as cytokines, can facilitate pathogen-mimetic immune cell activation. Finally, the nanoscale size range, which may be tunable, allows for multivalent presentation of antigenic and co-stimulatory moieties while facilitating receptor-mediated endocytosis via a wide variety of immune cells. These factors can be combined by stepwise chemical/biochemical synthesis (synthetic particulate systems), derived from viral capsids (virus-like particles), or isolated from and/or engineered into acellular bacterial vesicles (outer membrane vesicles).

and has also been included in nanoparticle formulations to enhance DC maturation [34]. Functional, protective immunity against certain pathogens or diseases is contingent on the vaccine correctly biasing the resulting immune response. A TH1-biased, cell-mediated response is particularly effective in protecting against intracellular pathogens, while a TH2-biased humoral response is necessary for protection against most bacteria and multicellular pathogens [18]. Whereas standard aluminum-based adjuvants bias the immune system towards a TH2 response, particle-based vaccines can be specifically engineered to induce either TH1 or TH2 responses. Immunization with smaller particles results in higher secretion of IFN-g by murine spleen cells, favoring a TH1 response, whereas larger particles induce www.sciencedirect.com

higher production of IL-4 and favor a TH2 response [19,35]. This polarization of the immune response may be a direct result of the difference in routes of uptake, as mentioned earlier [25], or it may be a result of the triggering of certain key co-stimulatory signals. For example, TLR-2 stimulation induces DCs to produce TH2-associated cytokines, while triggering TLR-9 results in the secretion of TH1-associated cytokines [22,36]. Furthermore, routes of administration and dosing also influence development of certain immune responses. For example, TH1-biased immunity seems to be most strongly stimulated via intralymphatic administration, where the environment is ideal for the induction of strong immune responses. In contrast, activation of the TH2 pathway appears to be independent of the route of administration [37]. Current Opinion in Biotechnology 2014, 28:51–58

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Overall, particle size engineering and the intentional incorporation of immunostimulatory molecules serves to increase the pathogen-like nature of nanoparticles by mimicking the pathogens themselves on a holistic, molecular level. By copying the immunologically relevant characteristics of pathogens, synthetic PLPs offer a safer and more effective alternative to the use of live attenuated or inactivated pathogens as vaccines. Significant advances have been made in engineering these particles to better resemble natural pathogens while also ensuring that the desired immune response is generated. However, variability in efficacy due to nuanced aspects of formulation, route of administration, and size suggest that more research is needed to better determine and optimize the role each of these factors play in stimulating immune responses.

Vaccine formulations based on the VLP technology have already come to market. In 1986, the VLP-based Recombivax HB1 was approved as the first recombinantly produced vaccine against hepatitis B. In more recent years, two VLP vaccines against HPV have been approved for use in the United States, Gardasil1 in 2006 and Cervarix1 in 2009 [43,49]. Numerous other VLP-based vaccines have been approved for veterinary use or are in clinical trials, demonstrating the versatility of VLPs and their future in vaccine development [50,51]. Of particular interest is the development of GlaxoSmithKline’s malaria vaccine called Mosquirix (RTS,S), wherein the antigen is delivered via hepatitis B capsid VLP [45]. Phase 3 trials have yielded promising results in terms of the vaccine’s ability to provide protection against the parasite [52].

Virus-like particle vaccines

Successes such as Gardasil1 have opened up the vaccine market to VLP-based formulations. However, as researchers begin to tackle more complex viral pathogens, VLPs will likely need to become more sophisticated as well. The increased corresponding cost of more sophisticated expression systems, as well as the inherent challenges associated with viral protein mutagenesis and modification, stand as significant barriers to such development. Design of the next generation of VLPs will undoubtedly be a nontrivial task, and future advances will likely focus on the engineering of more refined VLPs against increasingly challenging targets.

Virus-like particles (VLPs) are a promising alternative to live attenuated or inactivated virus vaccines commonly used for protection against viral diseases. At their most basic, VLPs are formed from the self-assembly of viral capsid proteins into particles that closely mimic the structure of natural virus particles. However, unlike viral particles, VLPs lack genomic material and thus are not susceptible to reversion [38]. Like synthetic pathogenmimetic particulate systems, VLPs are able to deliver their antigen loads efficiently to professional APCs as well as stimulate both cell-mediated and humoral immune responses at levels equal to or greater than existing formulations of protein subunits or inactivated viruses [39–41]. Various studies have shown that VLPs extend protective immunity against viral disease targets such as influenza, hand-foot-and-mouth disease, hepatitis B, and human papilloma virus (HPV) [39,42,43]. Lack of reversion and strong immune response also make VLPs a viable vaccine candidate to treat, rather than just suppress, the HIV-1 virus [44]. To make VLP vaccines, a single viral capsid protein is minimally required for the self-assembly of the viral particle. More complex VLPs require the co-expression of multiple capsid proteins to better mimic the properties of more complex viruses such as the bluetongue virus [38]. Additionally, viral particles may be used as a platform to deliver heterologous antigens. Examples include VLPs created from both simian and human immunodeficiency viral proteins and VLPs created to carry non-viral antigens [44,46]. VLPs are commonly produced in insect and yeast cell-based systems. These systems are favored for their ease of production, their ability to produce complex viral protein targets, and their capacity to be scaled up for commercial vaccine manufacturing [47,48]. Other systems have also been explored as potential VLP production platforms, including bacteria, mammalian and plant cells, and cell-free protein synthesis (CFPS) [49]. Current Opinion in Biotechnology 2014, 28:51–58

Outer membrane vesicle vaccines Outer membrane vesicles (OMVs), first documented almost 50 years ago, are naturally occurring proteoliposomes that bud from Gram-negative bacteria [53]. These vesicles reach sizes in the range of 50–250 nm and contain a single lipid bilayer encasing electron dense material, which is thought to correspond to the bacterial periplasm [54]. After the discovery that OMVs contain a variety of immunoactive virulence factors, researchers became interested in their potential to be used as vaccines [53]. Indeed, OMVs were quickly found to stimulate broadly protective humoral and mucosal immune responses, independent of additional commercial adjuvants or pathogenic components, against the bacterium from which they were isolated [55]. Apart from their ability to promote an effective immune response, OMVs have additional characteristics that make them viable vaccine candidates. First, like synthetic nanoparticle vaccines, they are classified as acellular, making them attractive replacements for live attenuated or inactivated pathogen vaccines [56]. Second, OMVs are able to present protein antigens in their native conformations [57], which is hypothesized to be important for effective antibody production [58]. Third, bacterial vesicles have been shown to be stable after long-term storage at 58C, which is an essential aspect of commercial viability [56,57]. www.sciencedirect.com

Pathogen-like particle vaccines Rosenthal et al. 55

With regards to clinical application, meningococcal OMV vaccines have been in use since the 1980s [57] and have shown particular utility in countering epidemics of Neisseria meningitidis serogroup B (MenB) [59]. In fact, until 2010 OMVs were the only commercially approved vaccine strategy against MenB [58]. Since then, Europe has approved 4CMenB or Bexsero1 (Novartis), a multiantigen vaccine that contains OMVs as one of the four components [60,61]. In addition to N. meningitidis serogroup B, other vesicle-producing bacteria have been studied to characterize the effects of their OMVs towards inducing protective immunity in humans. Some of these isolated OMVs have been shown to activate components of the immune system and stimulate protective immunity in mice, such as vesicles derived from Acinetobacter baumannii [62], Brucella melitensis [63], Francisella novicida [64], nontypeable Haemophilus influenzae [65], Salmonella typhimurium [66], Vibrio cholerae [67], and multiple strains of Shigellae [68]. It is generally accepted that protective antibodies are generated against one or a few dominant antigens located on the vesicle membrane surface [53]. For example, the major contributor to the T cell response generated by MenB OMVs is the outer membrane protein PorA. Unfortunately, the PorA protein varies widely between strains of MenB, meaning that isolated OMVs induce strainspecific immunity [57]. Therefore, to expand the application of these vesicles against pathogenic bacteria, wildtype OMVs were re-engineered at the genetic and molecular level. These recombinant approaches were used by the Netherlands Vaccine Institute to engineer a single strain that could produce nine of the antigenically different PorA variants, which they estimated would cover 75% of the major strains that affect humans around the world [57]. These studies realized the possibility of engineering unique, multi-antigen vaccines against this pathogen. The most crucial breakthrough in adapting OMVs into generalized PLP vaccines has come from their recent adaptation into heterologous antigen carriers. In this regard, the ability to remodel the outer surface of Escherichia coli-derived OMVs with a variety of recombinant antigens served as an important first step [69]. Shortly thereafter, it was demonstrated that recombinant antigens displayed on engineered OMVs derived from hypervesiculating E. coli were capable of eliciting strong antibody titers in immunized mice [70]. Along similar lines, a hypervesiculating S. typhimurium strain was engineered to produce a pneumococcal protein derivative in its periplasm, resulting in OMVs that successfully protected against a murine Streptococcus pneumonia challenge [71]. These examples of OMV engineering are founded on recombinant DNA technology in genetically tractable bacteria: modified protein antigens are cloned into plasmids that are introduced into the host strain by transformation, and the resulting protein fusions are expressed www.sciencedirect.com

in OMVs that are easily isolated from host cells. Hence, engineered OMVs represent a robust, tunable, and potentially cost-effective PLP vaccine platform. The use of OMVs as vaccines is not without its challenges. Lipopolysaccharide (LPS) is a major component of OMVs, which raises the potential issue of residual endotoxicity of the platform. Even though research has shown that LPS in a membrane, such as is found in OMVs, is 100 times less toxic than purified LPS [57], there is still ample motivation to adapt OMV production protocols to further detoxify OMV LPS. For example, detergent extraction is a popular method for isolating OMVs because it decreases LPS content [55,61]. Other methods involve structurally modifying the toxic lipid A to less harmful derivatives by genetically modifying the OMV-producing host strain. Specific examples include introducing exogenous genes, such as pagL [56], or mutating/deleting endogenous genes, such as msbB [72]. While resolving such challenges is nontrivial, OMVs continue to demonstrate great promise as a next-generation PLP vaccine platform that has the potential to seamlessly combine the biomimicry of VLPs with flexible, heterologous antigen integration and broad bacterial self-adjuvancy.

Conclusion The use of nanoparticulate carriers to enhance the efficacy of vaccination is a longstanding concept in the vaccine community, and recent advances in synthetic and biologically derived pathogen mimicry hold great promise in realizing the technology’s potential. A variety of vaccine applications have already benefited from the PLP technologies discussed here. For example, DNA vaccines, which have long suffered from the challenges of targeted delivery, have seen their efficacy and potency improve when combined with PLP technology [73–78]. Cancer vaccines, which have to contend with a number of immunological barriers inherent to a tumor’s engagement of local suppressive immunity, have similarly benefited as a result of PLP platforms [79,80–82]. Finally, PLP vaccines have shown impressive potency for pathogens that require targeted, biased immunostimulation across a variety of serotypes or life cycle stages to generate protective immunity [83,84,85,86,87,88]. Of course, PLP vaccine technology is not without certain challenges. As with all adjuvanting substances, there are questions concerning long-term safety. More complicated delivery vehicles often mean more costly syntheses, though for certain PLP technologies, like OMVs, it may be possible to increase the sophistication of delivery platform biomimicry without sacrificing vaccine economy. Regardless, the past several years have demonstrated that vaccine engineering is moving in a promising new direction with its manifold approaches to PLP-based vaccine delivery. By embracing the middle ground between recombinant molecular identity and whole pathogen vaccines, PLP Current Opinion in Biotechnology 2014, 28:51–58

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technology could potentially open the door to a new generation of potent, safe, and economical vaccination.

Acknowledgements JAR gratefully acknowledges Hertz Foundation and NSF graduate fellowship support. This work was supported by NSF CBET Award # 1264701 and NSF DGE Award # 1011509 (both to MPD).

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