Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation.

For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer-dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.

[Quantitative analysis of nine types of virus-like particles in human papilloma virus bulk by size-exclusion chromatography].

Cervical cancer is the fourth most common cancer among women. Human papilloma virus (HPV) is the most common cause of cervical cancer which accounts for 5% of all human cancers and results in about 528000 cases and 266000 deaths every year. HPV vaccines are considered the most effective strategy for the prevention of HPV infection and cervical carcinoma. Since 2006, three prophylactic vaccines against HPV have been available on the market, including bivalent vaccines, quadrivalent vaccines, and nine-valent vaccines. Among them, nine-valent vaccines have been reported to be the most effective. They can prevent 97% of the high-grade pre-cancer lesions. Virus-like particles (VLPs), which are arranged as 360 copies of capsid proteins L1, are the only antigens of the HPV vaccine. Nine-valent HPV vaccines are prepared by mixing nine types of VLPs with adjuvants. Thus, the quality of the VLPs, including their stability and content in the HPV bulk, is very important for developing HPV vaccines. In this study, a method was developed for the determination of the nine types of VLPs (HPV6/11/16/18/31/33/45/52/58) in HPV bulk by size exclusion chromatography (SEC). The parameters of this method were optimized in terms of column brand, pore size of stationary phase particles, buffer concentration, and pH value. SHIMSEN Ankylo SEC-300 column (300 mm×7.8 mm, 3 µm) combined with a buffer aqueous solution containing 300 mmol/L NaCl and 50 mmol/L phosphate (pH 7.0) was utilized to separate the VLPs from the matrix since a narrow peak shape and good repeatability for VLPs could be obtained with this column and mobile phase. The optimized method had a wide linear range, good repeatability (RSDs of peak area were not more than 5.0%), and a satisfactory sensitivity (LOQs in the range of 4.58-15.24 µg/mL). The optimized method was used to determine the VLPs in the HPV bulk. The LOQs of the current method were much lower than the content of the nine types of VLPs in the HPV bulk, indicating that this method was sensitive enough for the determination of the nine types of VLPs in the HPV bulk. The method was also used to determine the VLPs in an HPV bulk that had been stored at 4 ℃ for one week. A decrease in the nine types of VLPs in the range of 10.0%-62.6% was observed after they were stored at 4 ℃ for one week. An HPV vaccine was prepared by mixing the VLPs with an adjuvant. Thereafter, the VLPs were adsorbed on the surface of the adjuvant. The developed method was applied to determine the free VLPs in twelve batches of HPV vaccines from three different manufacturers. No obvious free protein was detected in the twelve batches of the HPV vaccines from the three manufacturers, indicating that VLPs from these manufactures react well with their aluminum adjuvant. Folin-phenol (Lowry assay) is commonly used for the determination of proteins in vaccines. It is based on the reduction of phosphomolybdotungstic mixed acid chromogen in the phosphomolybdotungstic reagent, which results in an absorbance maximum at 650 nm. The Lowry method was sensitive to interfering substances. Most interfering substances caused a lower color yield, while some detergents caused a slight increase in color. To reduce the effect of the interfering substances, a procedure for precipitating the proteins was usually required before the sample was tested. Thus, the Lowry assay is complex, time-consuming, and of low selectivity. Compared to the Lowry method, the method we developed is simpler and more automatic. It is a high-throughput method of determining VLPs. It can be used to determine VLPs in HPV bulk and free VLPs in HPV vaccines.

Caracterização imunológica da formulação vacinal baseada em VLP (Virus Like Particle) da Proteína Circumsporozoíta de Plasmodium vivax / Immunological characterization of the VLP (Virus Like Particle) vaccine formulation fused to Circumsporozoite Protein of Plasmodium vivax

O Plasmodium vivax é a espécie mais comum de parasita causador da malária humana encontrada fora da África, com maior endemicidade na Ásia, América Central e do Sul e Oceania. Embora o Plasmodium falciparum cause a maioria do número de mortes, o P. vivax pode levar à malária grave e resultar em morbimortalidade significativa. O desenvolvimento de uma vacina protetora será um passo importante para a eliminação da malária. Recentemente, uma formulação contendo as três variantes alélicas da proteína circumsporozoíta de P. vivax (PvCSP - All epitopes) induziu proteção parcial em camundongos após desafio com esporozoíto híbrido Plasmodium berghei (Pb), no qual as repetições centrais do PbCSP foram substituídas por repetições PvCSP-VK210 (esporozoítos Pb/Pv). No presente estudo, a proteína quimérica PvCSP contendo as variantes alélicas (VK210, VK247 e P. vivax-like) fusionadas com a proteína de nucleocapsídeo do vírus da caxumba (formando partículas semelhantes a nucleocapsídeos ou do inglês, NLP - Núcleo Like Particles) na ausência (NLP-CSPR) ou na presença do domínio C-terminal (CT) conservado da PvCSP (NLP-CSPCT). Para a realização do estudo selecionamos os adjuvantes Poly (I:C), um RNA sintético de dupla fita, agonista do receptor Toll do tipo 3 (TLR3) ou o adjuvante Montanide ISA 720, uma emulação óleo em agua. Para obter uma forte resposta imune, a levedura Pichia pastoris foi usada para expressar as proteínas recombinantes na forma de NLPs. Camundongos foram imunizados com cada uma das proteínas recombinantes em combinação com os adjuvantes citados. Embora ambas as NLPs tenham sido capazes de gerar uma forte resposta imune, com altos níveis de títulos e longevidade, apenas a formulação contendo a proteína NLP-CSPCT na presença do adjuvante Poly (I:C) foi selecionada para ser explorada em experimentos futuros. Esta proteína em combinação com o adjuvante Poly (I:C) induziu alta frequência de células secretoras de anticorpos específicas para o antígeno homólogo nos dias 5 e 30, no baço e na medula óssea, respectivamente. Altos títulos de IgG contra as 3 variantes de PvCSP foram detectados nos soros. Posteriormente camundongos imunizados com NLP-CSPCT foram desafiados com esporozoítos Pb/Pv e a parasitemia no 5º dia demonstrou proteção estéril em 30% dos camundongos desafiados. Portanto, a formulação vacinal gerada neste estudo tem potencial para ser explorada no desenvolvimento de uma vacina universal contra a malária causada por P. vivax

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP--All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein (assembling into nucleo like particles - NLP) in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To carry out the study, we selected the adjuvants Poly (I:C), a synthetic double-stranded RNA, Toll-like receptor 3 (TLR3) agonist or Montanide ISA 720 adjuvant, an oil-water emulation. To elicit stronger immune response, Pichia pastoris yeast was used to produce the NLPs. Mice were immunized with each recombinant protein in combination with above. Although both NLPs were able to generate stronger immune response, with high antibodies titer levels and longevity, formulation containing NLP-CSPCT in the presence of Poly (I:C) was selected to be explored in future experiments. NLP-CSPCT with Poly (I:C) adjuvant presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.

Integrating nanoparticle quantification and statistical design of experiments for efficient HIV-1 virus-like particle production in High Five cells.

The nature of enveloped virus-like particles (VLPs) has triggered high interest in their application to different research fields, including vaccine development. The baculovirus expression vector system (BEVS) has been used as an efficient platform for obtaining large amounts of these complex nanoparticles. To date, most of the studies dealing with VLP production by recombinant baculovirus infection utilize indirect detection or quantification techniques that hinder the appropriate characterization of the process and product. Here, we propose the application of cutting-edge quantification methodologies in combination with advanced statistical designs to exploit the full potential of the High Five/BEVS as a platform to produce HIV-1 Gag VLPs. The synergies between CCI, MOI, and TOH were studied using a response surface methodology approach on four different response functions: baculovirus infection, VLP production, VLP assembly, and VLP productivity. TOH and MOI proved to be the major influencing factors in contrast with previous reported data. Interestingly, a remarkable competition between Gag VLP production and non-assembled Gag was detected. Also, the use of nanoparticle tracking analysis and flow virometry revealed the existence of remarkable quantities of extracellular vesicles. The different responses of the study were combined to determine two global optimum conditions, one aiming to maximize the VLP titer (quantity) and the second aiming to find a compromise between VLP yield and the ratio of assembled VLPs (quality). This study provides a valuable approach to optimize VLP production and demonstrates that the High Five/BEVS can support mass production of Gag VLPs and potentially other complex nanoparticles.

Structural analysis of influenza vaccine virus-like particles reveals a multicomponent organization.

Influenza virus continues to be a major health problem due to the continually changing immunodominant head regions of the major surface glycoprotein, hemagglutinin (HA). However, some emerging vaccine platforms designed by biotechnology efforts, such as recombinant influenza virus-like particles (VLPs) have been shown to elicit protective antibodies to antigenically different influenza viruses. Here, using biochemical analyses and cryo-electron microscopy methods coupled to image analysis, we report the composition and 3D structural organization of influenza VLPs of the 1918 pandemic influenza virus. HA molecules were uniformly distributed on the VLP surfaces and the conformation of HA was in a prefusion state. Moreover, HA could be bound by antibody targeting conserved epitopes in the stem region of HA. Taken together, our analysis suggests structural parameters that may be important for VLP biotechnology such as a multi-component organization with (i) an outer component consisting of prefusion HA spikes on the surfaces, (ii) a VLP membrane with HA distribution permitting stem epitope display, and (iii) internal structural components.

Establishment of a yeast-based VLP platform for antigen presentation.

BACKGROUND: Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field. RESULTS: In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C. CONCLUSIONS: The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

Monitoring the Disassembly of Virus-like Particles by 19F-NMR.

Virus-like particles (VLPs) are stable protein cages derived from virus coats. They have been used extensively as biomolecular platforms, e.g., nanocarriers or vaccines, but a convenient in situ technique is lacking for tracking functional status. Here, we present a simple way to monitor disassembly of 19F-labeled VLPs derived from bacteriophage Qß by 19F NMR. Analysis of resonances, under a range of conditions, allowed determination not only of the particle as fully assembled but also as disassembled, as well as detection of a degraded state upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems.

Downstream processing of virus-like particles: single-stage and multi-stage aqueous two-phase extraction.

The demand for vaccines against untreated diseases has enforced the research and development of virus-like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, yeast and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction (ATPE). The benefits of ATPE have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of polyethylene (PEG) salt aqueous two-phase systems with a high capacity of up to 4.1mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of PEG, the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage ATPE on a centrifugal partition chromatography (CPC) device in 500mL scale. While single-stage ATPE enabled a DNA clearance of 99.6%, multi-stage ATPE improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage ATPE to 69.1% (40.1% VLP recovery) for the multi-stage ATPE. An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%.

Development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a Chikungunya virus-like particle vaccine.

To effectively support the development of a Chikungunya (CHIKV) virus-like particle (VLP) vaccine, a sensitive and robust high-performance liquid chromatography (HPLC) method that can quantitate CHIKV VLPs and monitor product purity throughout the manufacturing process is needed. We developed a sensitive reversed-phase HPLC (RP-HPLC) method that separates capsid, E1, and E2 proteins in CHIKV VLP vaccine with good resolution. Each protein component was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). The post-translational modifications on the viral glycoproteins E1 and E2 were further identified by intact protein mass measurements with liquid chromatography-mass spectrometry (LC-MS). The RP-HPLC method has a linear range of 0.51-12 µg protein, an accuracy of 96-106% and a precision of 12% RSD, suitable for vaccine product release testing. In addition, we demonstrated that the RP-HPLC method is useful for characterizing viral glycoprotein post-translational modifications, monitoring product purity during process development and assessing product stability during formulation development.

Seudopartículas virales como vacunas víricas en veterinaria / Virus-like particle-based vaccines for animal viral infections

La vacunación constituye uno de los procedimientos más eficaces para controlar los patógenos y prevenir enfermedades tanto en seres humanos como en el campo veterinario. Las vacunas tradicionales frente a enfermedades animales se basan por lo general en la utilización de virus atenuados o inactivados. Sin embargo, las vacunas de subunidad están ganando terreno progresivamente en el campo de la sanidad animal. Entre ellas, las vacunas basadas en pseudopartículas virales o VLPs (por su nombre en inglés virus-like particles), representan una de las estrategias más atractivas actualmente en el campo de las vacunas para animales. Las VLPs son estructuras proteicas con una geometría y uniformidad muy definidas, que mimetizan la estructura de los virus nativos pero carecen de genoma viral. Por lo general son antigénicamente indistinguibles de los virus de los que proceden y su empleo como inmunógenos presenta importantes ventajas en términos de seguridad. Las VLPs pueden inducir una fuerte respuesta inmune, tanto humoral como celular, y se ha demostrado que poseen capacidad de actuar como adyuvantes (self-adjuvanting). Además de su idoneidad como vacunas frente al virus homólogo del cual proceden, las VLPs también se pueden utilizar como vectores para la presentación multimérica de antígenos heterólogos. Las VLPs han mostrado una elevada eficacia como candidatos vacunales, sin embargo, hasta el momento sólo una vacuna basada en VLPs ha sido autorizada y comercializada en el campo veterinario. En este trabajo se revisa el estado actual de las VLP empleadas como nuevas estrategias vacunales en el campo de la veterinaria, analizando las potenciales ventajas y desafíos que enfrenta esta tecnología (AU)

Vaccination is considered one of the most effective ways to control pathogens and prevent diseases in humans as well as in the veterinary field. Traditional vaccines against animal viral diseases are based on inactivated or attenuated viruses, but new subunit vaccines are gaining attention from researchers in animal vaccinology. Among these, virus-like particles (VLPs) represent one of the most appealing approaches opening up interesting frontiers in animal vaccines. VLPs are robust protein scaffolds exhibiting well-defined geometry and uniformity that mimic the overall structure of the native virions but lack the viral genome. They are often antigenically indistinguishable from the virus from which they were derived and present important advantages in terms of safety. VLPs can stimulate strong humoral and cellular immune responses and have been shown to exhibit self-adjuvanting abilities. In addition to their suitability as a vaccine for the homologous virus from which they are derived, VLPs can also be used as vectors for the multimeric presentation of foreign antigens. VLPs have therefore shown dramatic effectiveness as candidate vaccines; nevertheless, only one veterinary VLP-base vaccine is licensed. Here, we review and examine in detail the current status of VLPs as a vaccine strategy in the veterinary field, and discuss the potential advantages and challenges of this technology (AU)

Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles.

The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs.

Microbial production of virus-like particle vaccine protein at gram-per-litre levels.

This study demonstrates the feasibility of large-scale production of murine polyomavirus VP1 protein in recombinant Escherichia coli as pentamers which are able to subsequently self-assemble in vitro into virus-like particles (VLPs). High-cell-density pH-stat fed-batch cultivation was employed to produce glutathione-S-transferase (GST)-VP1 fusion protein in soluble form. The expression of recombinant VP1 was induced with IPTG at different cell optical densities (OD at 600 nm of 20, 60 or 100). GST-VP1 production was highest when the culture was induced at a cell density of OD 60, with volumetric yield reaching 4.38 gL⁻¹ in 31h, which we believe is the highest volumetric productivity for viral capsid protein reported to date. The induction cell density is shown to have a significant effect on the overall volumetric yield of recombinant VP1 and on final cell density, but not on VLP quality. VP1 yield was enhanced 15-fold by scaling-up from shake flask to pH-stat fed-batch cultivation in a bioreactor. Although numerous studies have expressed structural viral protein in E. coli, we believe this is the first report of translation to bioreactors yielding gram-per-litre levels. This VLP production technology overcomes major drawbacks associated with eukaryotic cell-based vaccine production technologies, and propounds the scope for large-scale commercially viable E. coli based VLP production by significantly reducing vaccine production time and cost.