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This work presents a novel application of additive manufacturing in the design of self-assembling helical viral capsids using 3D-printed components. Expanding on prior work with 3D-printed self-assembling spherical capsids, we developed helical models that integrate geometric parameters and magnetic interactions to mimic key features of the assembly process of helical viral capsids. Using dual-helix phyllotactic patterns and simplified electrostatic simulations, these models consistently self-assemble into a cylinder, providing unique insights into the structural organization and stability of helical capsids. This accessible 3D-printed approach demonstrates the potential of additive manufacturing for research in mesoscale self-assembling models and in the education of complex biological assembly processes, promoting hands-on exploration of viral architecture and self-assembly mechanisms.

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 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.

•Design of influenza hemagglutinin antigen for display on modular capsomere.•Capsomere platform engineered for large antigen presentation.•E. coli-produced modular capsomere comprising HA1 highly immunogenic in chickens. 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.

•Silica nanoparticles were used to adjuvant sub-unit protein antigen.•Nanoparticles boosted antigen immune response without protein conjugation.•Non-carrier nanoparticles adjuvanting efficacy depends on particle size. 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 (50nm) but not for larger (1000nm) 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.

•Investigation of a modular capsomere vaccine presenting multiple M2e elements.•Protective efficacy demonstrated.•Rapid matching of broadly cross-protecting epitope to threat virus.•Extends coverage and possible impact of broadly cross-protecting eptiopes.•Synergy of manufacturing ease and protection opens new opportunities. 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 105 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.

•Modular capsomeres were prepared by non-chromatographic approaches.•Adjuvanted modular capsomeres induced high antibody titer after only a single shot.•“Goldilocks” level of contaminants provided a significant adjuvanting effect. 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 80EU of endotoxin per dose was inferior to highly purified and adjuvanted cCapM2e (2EU 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.

•Purified L1 protein expressed in E. coli self-assemble into capsomeres and VLP.•Recombinant L1 protein is thermostable and has no mutagenic risk.•Vaccinated cattle shown significant humoral response against recombinant L1 protein. Bovine papillomatosis is an infectious disease that is caused by bovine papillomavirus (BPV), which results in important economic losses. However, no BPV vaccines or effective treatment methods are commercially available to date. Moreover, the absence of papillomavirus replication in vitro makes the use of recombinant protein a promising candidate for vaccine formulations. Hence, we developed an integrated study on the L1 capsid protein of BPV-1, obtained from a bacterial expression system, regarding its purification, biosafety, thermostability and immunogenicity. The results indicated an absence of genotoxicity of the purified recombinant L1 protein, β-sheet prevalence of secondary structure folding, protein stability under high temperatures as well as the presence of capsomeres and VLPs. In addition, preliminary experimental vaccination of calves showed the production of specific antibodies against BPV-1 L1.

Immunization with L1 as pentavalent capsomeres or virus-like particles (VLPs) generates high and long-lived titers of neutralizing antibodies and protection primarily against the human papillomavirus (HPV) type from which the vaccine was derived. Conversely, vaccination with L2 minor capsid protein derived from multiple HPV types induces lower titer, but more broadly neutralizing and protective antibody responses. We combined the advantages of each protective antigen by immunization with titrated doses of multi-type L2 with either L1 capsomeres or VLP. We observed no significant interference between the L1 and L2 antibody response upon co-administration of L1 vaccines with multi-type L2 vaccines.

HPV 16 L1 capsomeres purified from Escherichia coli represent a promising and potentially cost-effective alternative to the recently licensed VLP-based vaccines for the prevention of cervical cancer. However, recombinant protein preparations from bacteria always bear the risk of contaminating endotoxins which are highly toxic in humans and therefore have to be eliminated from vaccine preparations. In this study, we measured the LPS concentration at various stages of the purification of HPV 16 L1 from E. coli and determined that it enhances the immunogenicity of HPV 16 VLPs and capsomeres. We confirmed the immunogenicity of the L1 capsomeres in TLR4 −/− mice without the enhancing effect of the LPS and then elaborated a suitable protocol using Triton X-114 phase separation for the removal of LPS without any significant protein loss or influence on the structural integrity of the particles. The LPS-free capsomeres purified from E. coli induced neutralizing L1-specific antibodies. Our results demonstrate the excellent potential of capsomeres as an economically interesting alternative vaccine to prevent cervical cancer that could be made available in developing countries.

Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T₀, T₁ and T₂ generations. Taken together, these results indicate the possibility of producing a low-cost capsomere-based vaccine by plastids.