Chimeric Virus-Like Particles and Capsomeres Induce Similar CD8+ T Cell Responses but Differ in Capacity to Induce CD4+ T Cell Responses and Antibody Responses.

Despite extensive research, the development of an effective malaria vaccine remains elusive. The induction of robust and sustained T cell and antibody response by vaccination is an urgent unmet need. Chimeric virus-like particles (VLPs) are a promising vaccine platform. VLPs are composed of multiple subunit capsomeres which can be rapidly produced in a cost-effective manner, but the ability of capsomeres to induce antigen-specific cellular immune responses has not been thoroughly investigated. Accordingly, we have compared chimeric VLPs and their sub-unit capsomeres for capacity to induce CD8+ and CD4+ T cell and antibody responses. We produced chimeric murine polyomavirus VLPs and capsomeres each incorporating defined CD8+ T cell, CD4+ T cell or B cell repeat epitopes derived from Plasmodium yoelii CSP. VLPs and capsomeres were evaluated using both homologous or heterologous DNA prime/boost immunization regimens for T cell and antibody immunogenicity. Chimeric VLP and capsomere vaccine platforms induced robust CD8+ T cell responses at similar levels which was enhanced by a heterologous DNA prime. The capsomere platform was, however, more efficient at inducing CD4+ T cell responses and less efficient at inducing antigen-specific antibody responses. Our data suggest that capsomeres, which have significant manufacturing advantages over VLPs, should be considered for diseases where a T cell response is the desired outcome.

Chimeric Murine Polyomavirus Virus-Like Particles Induce Plasmodium Antigen-Specific CD8+ T Cell and Antibody Responses.

An effective vaccine against the Plasmodium parasite is likely to require the induction of robust antibody and T cell responses. Chimeric virus-like particles are an effective vaccine platform for induction of antibody responses, but their capacity to induce robust cellular responses and cell-mediated protection against pathogen challenge has not been established. To evaluate this, we produced chimeric constructs using the murine polyomavirus structural protein with surface-exposed CD8+ or CD4+ T cell or B cell repeat epitopes derived from the Plasmodium yoelii circumsporozoite protein, and assessed immunogenicity and protective capacity in a murine model. Robust CD8+ T cell responses were induced by immunization with the chimeric CD8+ T cell epitope virus-like particles, however CD4+ T cell responses were very low. The B cell chimeric construct induced robust antibody responses but there was no apparent synergy when T cell and B cell constructs were administered as a pool. A heterologous prime/boost regimen using plasmid DNA priming followed by a VLP boost was more effective than homologous VLP immunization for cellular immunity and protection. These data show that chimeric murine polyomavirus virus-like particles are a good platform for induction of CD8+ T cell responses as well as antibody responses.

Structural-based designed modular capsomere comprising HA1 for low-cost poultry influenza vaccination.

Highly pathogenic avian influenza (HPAI) viruses cause a severe and lethal infection in domestic birds. The increasing number of HPAI outbreaks has demonstrated the lack of capabilities to control the rapid spread of avian influenza. Poultry vaccination has been shown to not only reduce the virus spread in animals but also reduce the virus transmission to humans, preventing potential pandemic development. However, existing vaccine technologies cannot respond to a new virus outbreak rapidly and at a cost and scale that is commercially viable for poultry vaccination. Here, we developed modular capsomere, subunits of virus-like particle, as a low-cost poultry influenza vaccine. Modified murine polyomavirus (MuPyV) VP1 capsomere was used to present structural-based influenza Hemagglutinin (HA1) antigen. Six constructs of modular capsomeres presenting three truncated versions of HA1 and two constructs of modular capsomeres presenting non-modified HA1 have been generated. These modular capsomeres were successfully produced in stable forms using Escherichia coli, without the need for protein refolding. Based on ELISA, this adjuvanted modular capsomere (CaptHA1-3C) induced strong antibody response (almost 105endpoint titre) when administered into chickens, similar to titres obtained in the group administered with insect cell-based HA1 proteins. Chickens that received adjuvanted CaptHA1-3C followed by challenge with HPAI virus were fully protected. The results presented here indicate that this platform for bacterially-produced modular capsomere could potentially translate into a rapid-response and low-cost vaccine manufacturing technology suitable for poultry vaccination.

Modular virus-like particles for sublingual vaccination against group A streptococcus.

Infection with Group A streptococcus (GAS)-an oropharyngeal pathogen-leads to mortality and morbidity, primarily among developing countries and indigenous populations in developed countries. The development of safe and affordable GAS vaccines is challenging, due to the presence of various unique GAS serotypes, antigenic variation within the same serotype, and potential auto-immune responses. In the present study, we evaluated the use of a sublingual freeze-dried (FD) formulation based on immunogenic modular virus-like particles (VLPs) carrying the J8 peptide (J8-VLPs) as a potential safe and cost-effective GAS vaccine for inducing protective systemic and mucosal immunity. By using in vivo tracing of the sublingual J8-VLPs, we visualized the draining of J8-VLPs into the submandibular lymph nodes, in parallel with its rapid absorption into the systemic circulation, which support the induction of effective immune responses in both systemic and mucosal compartments. The sublingual administration of J8-VLPs resulted in a high serum IgG antibody level, with a good balance of Th1 and Th2 immune responses. Of note, sublingual vaccination with J8-VLPs elicited high levels of IgA antibody in the saliva. The co-administration of mucosal adjuvant cholera toxin (CT) further enhanced the increase in salivary IgA antibody levels induced by the J8-VLPs formulation. Moreover, the levels of salivary IgA and serum IgG observed following the administration of the CT-adjuvanted FD formulation of J8-VLPs (FD-J8-VLPs) and non-FD formulation of J8-VLPs were comparable. In fact, the saliva isolated from mice immunized with J8-VLPs and FD-J8-VLPs with CT demonstrated opsonizing activity against GAS in vitro. Thus, we observed that the sublingually delivered FD formulation of microbially produced modular VLPs could prevent and control GAS diseases in endemic areas in a cost-effective manner.

Non-chromatographic preparation of a bacterially produced single-shot modular virus-like particle capsomere vaccine for avian influenza.

Highly pathogenic avian influenza (HPAI) causes significant economic loss, reduced food security and poses an ongoing pandemic threat. Poultry vaccination significantly decreases these problems and recognizes that the health of humans, animals and ecosystems are connected. Low-cost manufacture of poultry vaccine matched quickly to the ever-changing circulating strain is needed for effective vaccination. Here, we re-engineered the process to manufacture bacterially synthesized modular capsomere comprising influenza M2e, previously shown to confer complete protection in challenged mice, for application in poultry. Modular capsomere was prepared using a simplified non-chromatographic salting-out precipitation method and its immunogenicity tested in vivo in poultry. Modular capsomere crudely purified by precipitation (pCapM2e) contained more contaminants than equivalent product purified by chromatography (cCapM2e). Unadjuvanted pCapM2e containing 80 EU of endotoxin per dose was inferior to highly purified and adjuvanted cCapM2e (2 EU per dose). However, addition of adjuvant to pCapM2e resulting in high immunogenicity after only a single dose of vaccination, yet without any local adverse reaction. This finding suggests a strong synergy between adjuvant, antigen and contaminants, and the possible existence of a "Goldilocks" level of contaminants, where high immunogenicity and low reactogenicity can be obtained in a single-shot vaccination. The simplified process offers potential cost and speed advantages to address the needs in influenza poultry vaccination in low-cost veterinary markets.

Non-carrier nanoparticles adjuvant modular protein vaccine in a particle-dependent manner.

Nanoparticles are increasingly used to adjuvant vaccine formulations due to their biocompatibility, ease of manufacture and the opportunity to tailor their size, shape, and physicochemical properties. The efficacy of similarly-sized silica (Si-OH), poly (D,L-lactic-co-glycolic acid) (PLGA) and poly caprolactone (PCL) nanoparticles (nps) to adjuvant recombinant capsomere presenting antigenic M2e modular peptide from Influenza A virus (CapM2e) was investigated in vivo. Formulation of CapM2e with Si-OH or PLGA nps significantly boosted the immunogenicity of modular capsomeres, even though CapM2e was not actively attached to the nanoparticles prior to injection (i.e., formulation was by simple mixing). In contrast, PCL nps showed no significant adjuvant effect using this simple-mixing approach. The immune response induced by CapM2e alone or formulated with nps was antibody-biased with very high antigen-specific antibody titer and less than 20 cells per million splenocytes secreting interferon gamma. Modification of silica nanoparticle surface properties through amine functionalization and pegylation did not lead to significant changes in immune response. This study confirms that simple mixing-based formulation can lead to effective adjuvanting of antigenic protein, though with antibody titer dependent on nanoparticle physicochemical properties.

Protective efficacy of a bacterially produced modular capsomere presenting M2e from influenza: extending the potential of broadly cross-protecting epitopes.

Influenza A viruses drift and shift, emerging as antigenically distinct strains that lead to epidemics and pandemics of varying severity. Even epitopes associated with broad cross-protection against different strains, such as the ectodomain of matrix protein 2 (M2e), mutate unpredictably. Vaccine protective efficacy is only ensured when the emerging virus lies within the vaccine's cross-protective domain, which is poorly defined in most situations. When virus emerges outside this domain it is essential to rapidly re-engineer the vaccine and hence re-center the cross-protective domain on the new virus. This approach of vaccine re-engineering in response to virus change is the cornerstone of the current influenza control system, based on annual prediction and/or pandemic reaction. This system could become more responsive, and perhaps preventative, if its speed could be improved. Here, we demonstrate vaccine efficacy of a rapidly manufacturable modular capsomere presenting the broadly cross-protecting M2e epitope from influenza. M2e inserted into a viral capsomere at the DNA level was expressed in Escherichia coli as a fusion protein (Wibowo et al., 2013). Immunization of mice with this modular capsomere adjuvanted with conventional aluminum hydroxide induced high (more than 10(5) endpoint titer) levels of M2e-specific antibodies that reduced disease severity and viral load in the lungs of challenged mice. The combination of rapid manufacturability of modular capsomere presented in this study, and the established cross-protective efficacy of M2e, allow rapid matching of vaccine to the circulating virus and hence rapid re-centering of the vaccine's cross-protective domain onto the virus. This approach synergizes the discussed benefits of broadly cross-protecting epitopes with rapid scale-up vaccine manufacture using microbial cell factories.

Co-administration of non-carrier nanoparticles boosts antigen immune response without requiring protein conjugation.

Nanotechnology promises a revolution in medicine including through new vaccine approaches. The use of nanoparticles in vaccination has, to date, focused on attaching antigen directly to or within nanoparticle structures to enhance antigen uptake by immune cells. Here we question whether antigen incorporation with the nanoparticle is actually necessary to boost vaccine effectiveness. We show that the immunogenicity of a sub-unit protein antigen was significantly boosted by formulation with silica nanoparticles even without specific conjugation of antigen to the nanoparticle. We further show that this effect was observed only for virus-sized nanoparticles (50 nm) but not for larger (1,000 nm) particles, demonstrating a pronounced effect of nanoparticle size. This non-attachment approach has potential to radically simplify the development and application of nanoparticle-based formulations, leading to safer and simpler nanoparticle applications in vaccine development.

Microbially synthesized modular virus-like particles and capsomeres displaying group A streptococcus hypervariable antigenic determinants.

Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 10(4) ). Second, delivery by capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 µg (corresponding to <600 ng of each GAS peptide), induced significant titers of IgGs specific to each peptide, demonstrating that a multivalent, broad-coverage VLP vaccine formulation was possible. In summary, the modular VLPs and capsomeres reported here demonstrate, with promising preliminary data, innovative ways to design GAS vaccines using VLP and capsomere delivery systems amenable to microbial synthesis, potentially adoptable by developing countries.

Bioengineering virus-like particles as vaccines.

Virus-like particle (VLP) technology seeks to harness the optimally tuned immunostimulatory properties of natural viruses while omitting the infectious trait. VLPs that assemble from a single protein have been shown to be safe and highly efficacious in humans, and highly profitable. VLPs emerging from basic research possess varying levels of complexity and comprise single or multiple proteins, with or without a lipid membrane. Complex VLP assembly is traditionally orchestrated within cells using black-box approaches, which are appropriate when knowledge and control over assembly are limited. Recovery challenges including those of adherent and intracellular contaminants must then be addressed. Recent commercial VLPs variously incorporate steps that include VLP in vitro assembly to address these problems robustly, but at the expense of process complexity. Increasing research activity and translation opportunity necessitate bioengineering advances and new bioprocessing modalities for efficient and cost-effective production of VLPs. Emerging approaches are necessarily multi-scale and multi-disciplinary, encompassing diverse fields from computational design of molecules to new macro-scale purification materials. In this review, we highlight historical and emerging VLP vaccine approaches. We overview approaches that seek to specifically engineer a desirable immune response through modular VLP design, and those that seek to improve bioprocess efficiency through inhibition of intracellular assembly to allow optimal use of existing purification technologies prior to cell-free VLP assembly. Greater understanding of VLP assembly and increased interdisciplinary activity will see enormous progress in VLP technology over the coming decade, driven by clear translational opportunity.

Nanoparticle vaccines.

Nanotechnology increasingly plays a significant role in vaccine development. As vaccine development orientates toward less immunogenic "minimalist" compositions, formulations that boost antigen effectiveness are increasingly needed. The use of nanoparticles in vaccine formulations allows not only improved antigen stability and immunogenicity, but also targeted delivery and slow release. A number of nanoparticle vaccines varying in composition, size, shape, and surface properties have been approved for human use and the number of candidates is increasing. However, challenges remain due to a lack of fundamental understanding regarding the in vivo behavior of nanoparticles, which can operate as either a delivery system to enhance antigen processing and/or as an immunostimulant adjuvant to activate or enhance immunity. This review provides a broad overview of recent advances in prophylactic nanovaccinology. Types of nanoparticles used are outlined and their interaction with immune cells and the biosystem are discussed. Increased knowledge and fundamental understanding of nanoparticle mechanism of action in both immunostimulatory and delivery modes, and better understanding of in vivo biodistribution and fate, are urgently required, and will accelerate the rational design of nanoparticle-containing vaccines.

Effects of pre-existing anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine.

Modularization of a peptide antigen for presentation on a microbially synthesized murine polyomavirus (MuPyV) virus-like particle (VLP) offers a new alternative for rapid and low-cost vaccine delivery at a global scale. In this approach, heterologous modules containing peptide antigenic elements are fused to and displayed on the VLP carrier, allowing enhancement of peptide immunogenicity via ordered and densely repeated presentation of the modules. This study addresses two key engineering questions pertaining to this platform, exploring the effects of (i) pre-existing carrier-specific immunity on modular VLP vaccine effectiveness and (ii) increase in the antigenic element number per VLP on peptide-specific immune response. These effects were studied in a mouse model and with modular MuPyV VLPs presenting a group A streptococcus (GAS) peptide antigen, J8i. The data presented here demonstrate that immunization with a modular VLP could induce high levels of J8i-specific antibodies despite a strong pre-existing anti-carrier immune response. Doubling of the J8i antigenic element number per VLP did not enhance J8i immunogenicity at a constant peptide dose. However, the strategy, when used in conjunction with increased VLP dose, could effectively increase the peptide dose up to 10-fold, leading to a significantly higher J8i-specific antibody titer. This study further supports feasibility of the MuPyV modular VLP vaccine platform by showing that, in the absence of adjuvant, modularized GAS antigenic peptide at a dose as low as 150 ng was sufficient to raise a high level of peptide-specific IgGs indicative of bactericidal activity.

A microbial platform for rapid and low-cost virus-like particle and capsomere vaccines.

Studies on a platform technology able to deliver low-cost viral capsomeres and virus-like particles are described. The technology involves expression of the VP1 structural protein from murine polyomavirus (MuPyV) in Escherichia coli, followed by purification using scaleable units and optional cell-free VLP assembly. Two insertion sites on the surface of MuPyV VP1 are exploited for the presentation of the M2e antigen from influenza and the J8 peptide from Group A Streptococcus (GAS). Results from testing on mice following subcutaneous administration demonstrate that VLPs are self adjuvating, that adding adjuvant to VLPs provides no significant benefit in terms of antibody titre, and that adjuvanted capsomeres induce an antibody titre comparable to VLPs but superior to unadjuvanted capsomere formulations. Antibodies raised against GAS J8 peptide following immunization with chimeric J8-VP1 VLPs are bactericidal against a GAS reference strain. E. coli is easily and widely cultivated, and well understood, and delivers unparalleled volumetric productivity in industrial bioreactors. Indeed, recent results demonstrate that MuPyV VP1 can be produced in bioreactors at multi-gram-per-litre levels. The platform technology described here therefore has the potential to deliver safe and efficacious vaccine, quickly and cost effectively, at distributed manufacturing sites including those in less developed countries. Additionally, the unique advantages of VLPs including their stability on freeze drying, and the potential for intradermal and intranasal administration, suggest this technology may be suited to numerous diseases where adequate response requires large-scale and low-cost vaccine manufacture, in a way that is rapidly adaptable to temporal or geographical variation in pathogen molecular composition.