RESUMO
BACKGROUND: A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body. RESULTS: In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group. CONCLUSION: Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.
Assuntos
Endotoxinas , Escherichia coli , Lipídeo A , Camundongos Endogâmicos BALB C , Parvovirus Suíno , Vacinas de Partículas Semelhantes a Vírus , Animais , Camundongos , Escherichia coli/genética , Escherichia coli/metabolismo , Parvovirus Suíno/imunologia , Parvovirus Suíno/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Endotoxinas/imunologia , Células RAW 264.7 , Lipídeo A/imunologia , Lipídeo A/análogos & derivados , Interleucina-6/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Feminino , Suínos , Lipopolissacarídeos/imunologiaRESUMO
Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Imunidade Celular , Imunidade Humoral , Vacina Antirrábica , Vírus da Raiva , Raiva , Vacinas de Partículas Semelhantes a Vírus , Animais , Vacina Antirrábica/imunologia , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/genética , Raiva/prevenção & controle , Raiva/imunologia , Vírus da Raiva/imunologia , Vírus da Raiva/genética , Camundongos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Feminino , Vacinas de mRNA/imunologia , Camundongos Endogâmicos BALB C , Nucleosídeos/imunologia , Glicoproteínas/imunologia , Glicoproteínas/genética , Humanos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/genética , Antígenos Virais/imunologia , Antígenos Virais/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , RNA Mensageiro/genética , RNA Mensageiro/imunologiaRESUMO
Intact capsids of foot-and-mouth disease virus (FMDV) play a vital role in eliciting a protective immune response. Any change in the physico-chemical environment of the capsids results in dissociation and poor immunogenicity. Structural bioinfomatics studies have been carried out to predict the amino acids at the interpentameric region that resulted in the identification of mutant virus-like particles(VLPs) of FMDV serotype Asia1/IND/63/1972. The insect cell expressed VLPs were evaluated for their stability by sandwich ELISA. Among 10 mutants, S93H showed maximum retention of antigenicity at different temperatures, indicating its higher thermal stability as revealed by the in-silico analysis and retained the antigenic sites of the virus demonstrated by Sandwich ELISA. The concordant results of the liquid phase blocking ELISA for estimation of antibody titre of known sera with stable mutant VLP as antigen in place of virus antigen demonstrate its diagnostic potential. The stable mutant VLP elicited a robust immune response with 85.6 % protection in guinea pigs against virus challenge. The stabilized VLP based antigen requires minimum biosafety and cold storage for production and transit besides, complying with differentiation of infected from vaccinated animals. It can effectively replace the conventional virus handling during antigen production for prophylactic and diagnostic use.
RESUMO
Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.
Assuntos
Capsídeo , Proteínas de Fluorescência Verde , Vírus da Hepatite B , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Capsídeo/química , Capsídeo/metabolismo , Montagem de Vírus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírion/metabolismo , Vírion/química , Propriedades de SuperfícieRESUMO
A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza.
Assuntos
Apresentação de Antígeno , Proteínas do Capsídeo , Vírus da Hepatite E , Nicotiana , Proteínas Recombinantes de Fusão , Proteínas da Matriz Viral , Vírus da Hepatite E/imunologia , Vírus da Hepatite E/genética , Nicotiana/virologia , Nicotiana/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Plantas Geneticamente Modificadas , Vírus da Influenza A/imunologia , Vírus da Influenza A/genética , Hepatite E/imunologia , Hepatite E/prevenção & controle , Hepatite E/virologia , Proteínas ViroporinasRESUMO
Cutaneous leishmaniasis (CL) is a tropical disease endemic in many parts of the world. Characteristic clinical manifestations of CL include the formation of ulcerative skin lesions that can inflict life-long disability if left untreated. Although drugs are available, they are unaffordable and out of reach for individuals who need them the most. Developing a highly cost-efficient CL vaccine could address this problem but such a vaccine remains unavailable. Here, we developed a chimeric influenza virus-like particle expressing the Leishmania amazonensis promastigote surface antigen (LaPSA-VLP). LaPSA-VLPs were self-assembled in Spodoptera frugiperda insect cell lines using the baculovirus expression system. After characterizing the vaccines and confirming successful VLP assembly, BALB/c mice were immunized with these vaccines for efficacy assessment. Sera acquired from mice upon subcutaneous immunization with the LaPSA-VLP specifically interacted with the L. amazonensis soluble total antigens. LaPSA-VLP-immunized mice elicited significantly greater quantities of parasite-specific IgG from the spleens, popliteal lymph nodes, and footpads than unimmunized mice. LaPSA-VLP immunization also enhanced the proliferation of B cell populations in the spleens of mice and significantly lessened the CL symptoms, notably the footpad swelling and IFN-γ-mediated inflammatory response. Overall, immunizing mice with the LaPSA-VLPs prevented mice from developing severe CL symptoms, signifying their developmental potential.
RESUMO
The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation.Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A1; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; µm: micrometer; µM: micromole.
RESUMO
Getah virus (GETV) is an emerging mosquito-borne virus with economic impact on the livestock industry in East Asia. In this study, we successfully produced GETV virus-like particles (VLPs) in insect cells using the baculovirus expression vector system. We show that the GETV envelope glycoproteins were successfully expressed at the surface of the insect cell and were glycosylated. VLPs were isolated from the culture fluid as enveloped particles of 60-80 nm in diameter. Two 1 µg vaccinations with this GETV VLP vaccine, without adjuvant, generated neutralizing antibody responses and protected wild-type C57/BL6 mice against GETV viremia and arthritic disease. The GETV VLP vaccine may find application as a horse and/or pig vaccine in the future.
RESUMO
Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage malaria vaccine antigen target, currently in a phase 2b clinical trial as a full-length soluble protein/adjuvant vaccine candidate called RH5.1/Matrix-M. We identify that disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees and that a re-engineered and stabilized immunogen (including just the alpha-helical core of RH5) induces a qualitatively superior growth inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this immunogen, termed "RH5.2," to hepatitis B surface antigen virus-like particles (VLPs) using the "plug-and-display" SpyTag-SpyCatcher platform technology also enables superior quantitative antibody immunogenicity over soluble protein/adjuvant in vaccinated mice and rats. These studies identify a blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in phase 1a/b clinical trials.
Assuntos
Anticorpos Antiprotozoários , Vacinas Antimaláricas , Plasmodium falciparum , Proteínas de Protozoários , Vacinas de Partículas Semelhantes a Vírus , Animais , Vacinas Antimaláricas/imunologia , Anticorpos Antiprotozoários/imunologia , Plasmodium falciparum/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Humanos , Camundongos , Proteínas de Protozoários/imunologia , Ratos , Malária Falciparum/prevenção & controle , Malária Falciparum/imunologia , Antígenos de Protozoários/imunologia , Feminino , Proteínas de Transporte/imunologia , Camundongos Endogâmicos BALB CRESUMO
Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.
Assuntos
Anticorpos Neutralizantes , Bombyx , Vírus da Dengue , Camundongos Endogâmicos BALB C , Proteínas do Envelope Viral , Animais , Bombyx/genética , Bombyx/virologia , Bombyx/metabolismo , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Camundongos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/biossíntese , Anticorpos Neutralizantes/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/biossíntese , Vacinas contra Dengue/imunologia , Vacinas contra Dengue/genética , Anticorpos Antivirais/imunologia , Dengue/imunologia , Dengue/virologia , Sorogrupo , Domínios Proteicos , FemininoRESUMO
RNA bacteriophage MS2-derived virus-like particles (VLPs) have been widely used in biomedical research as model systems to study virus assembly, structure-function relationships, vaccine development, and drug delivery. Considering the diverse utility of these VLPs, a systemic engineering approach has been utilized to generate smaller particles with optimal serum stability and tissue penetrance. Additionally, it is crucial to demonstrate the overall stability of these mini MS2 VLPs, ensuring cargo protection until they reach their target cell/organ. However, no detailed analysis of the thermal stability and heat-induced disassembly of MS2 VLPs has yet been attempted. In this work, we investigated the thermal stability of both wild-type (WT) MS2 VLP and its "mini" variant containing S37P mutation (mini MS2 VLP). The mini MS2 VLP exhibits a higher capsid melting temperature (Tm) when compared to its WT MS2 VLP counterpart, possibly attributed to its smaller interdimer angle. Our study presents that the thermal unfolding of MS2 VLPs follows a sequential process involving particle destabilization, nucleic acid exposure/melting, and disassembly of VLP. This observation underscores the disruption of cooperative intersubunit interactions and protein-nucleic acid interactions, shedding light on the mechanism of heat-induced VLP disassembly.
Assuntos
Levivirus , Levivirus/genética , Levivirus/química , Levivirus/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Temperatura , Mutação , Temperatura Alta , Vírion/metabolismo , Vírion/química , Vírion/genética , Capsídeo/metabolismo , Capsídeo/químicaRESUMO
Porcine circovirus type 4 (PCV4), a recently identified circovirus, is prevalent in numerous provinces in China, as well as in South Korea, Thailand, and Europe. PCV4 virus rescued from an infectious clone showed pathogenicity, suggesting the economic impact of PCV4. However, there remains a lack of understanding regarding the immunogenicity and epitopes of PCV4. This study generated a monoclonal antibody (MAb) 1D8 by immunizing mice with PCV4 virus-like particles (VLPs). Subsequently, the epitope recognized by the MAb 1D8 was identified by truncated protein expression and alanine scanning mutagenesis analysis. Results showed that the 225PKQG228 located at the C-terminus of the PCV4 Cap protein is the minimal motif binding to the MAb. Homology modeling analysis and immunoelectron microscopy revealed that the epitope extends beyond the outer surface of the PCV4 VLP. Moreover, the epitope is highly conserved among PCV4 strains and does not react with other PCVs. Together, the MAb 1D8 recognized epitope shows potential for detecting PCV4. These findings significantly contribute to the design of antigens for PCV4 detection and control strategies. IMPORTANCE: Porcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Proteínas do Capsídeo , Circovirus , Epitopos de Linfócito B , Circovirus/imunologia , Circovirus/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Animais , Anticorpos Monoclonais/imunologia , Camundongos , Suínos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Anticorpos Antivirais/imunologia , Camundongos Endogâmicos BALB C , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Doenças dos Suínos/virologia , Doenças dos Suínos/imunologia , FemininoRESUMO
Malaria is a global disease affecting a large portion of the world's population. Although vaccines have recently become available, their efficacies are suboptimal. We generated virus-like particles (VLPs) that expressed either apical membrane antigen 1 (AMA1) or microneme-associated antigen (MIC) of Plasmodium berghei and compared their efficacy in BALB/c mice. We found that immune sera acquired from AMA1 VLP- or MIC VLP-immunized mice specifically interacted with the antigen of choice and the whole P. berghei lysate antigen, indicating that the antibodies were highly parasite-specific. Both VLP vaccines significantly enhanced germinal center B cell frequencies in the inguinal lymph nodes of mice compared with the control, but only the mice that received MIC VLPs showed significantly enhanced CD4+ T cell responses in the blood following P. berghei challenge infection. AMA1 and MIC VLPs significantly suppressed TNF-α and interleukin-10 production but had a negligible effect on interferon-γ. Both VLPs prevented excessive parasitemia buildup in immunized mice, although parasite burden reduction induced by MIC VLPs was slightly more effective than that induced by AMA1. Both VLPs were equally effective at preventing body weight loss. Our findings demonstrated that the MIC VLP was an effective inducer of protection against murine experimental malaria and should be the focus of further development.
Assuntos
Antígenos de Protozoários , Vacinas Antimaláricas , Proteínas de Membrana , Plasmodium berghei , Proteínas de Protozoários , Vacinas de Partículas Semelhantes a Vírus , Animais , Feminino , Camundongos , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Malária/prevenção & controle , Malária/imunologia , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/administração & dosagem , Proteínas de Membrana/imunologia , Camundongos Endogâmicos BALB C , Parasitemia/imunologia , Parasitemia/prevenção & controle , Plasmodium berghei/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagemRESUMO
This work aimed to set inline Raman spectroscopy models to monitor biochemically (viable cell density, cell viability, glucose, lactate, glutamine, glutamate, and ammonium) all upstream stages of a virus-like particle-making process. Linear (Partial least squares, PLS; Principal components regression, PCR) and nonlinear (Artificial neural networks, ANN; supported vector machine, SVM) modeling approaches were assessed. The nonlinear models, ANN and SVM, were the more suitable models with the lowest absolute errors. The mean absolute error of the best models within the assessed parameter ranges for viable cell density (0.01-8.83 × 106 cells/mL), cell viability (1.3-100.0 %), glucose (5.22-10.93 g/L), lactate (18.6-152.7 mg/L), glutamine (158-1761 mg/L), glutamate (807.6-2159.7 mg/L), and ammonium (62.8-117.8 mg/L) were 1.55 ± 1.37 × 106 cells/mL (ANN), 5.01 ± 4.93 % (ANN), 0.27 ± 0.22 g/L (SVM), 4.7 ± 2.6 mg/L (SVM), 51 ± 49 mg/L (ANN), 57 ± 39 mg/L (SVM) and 2.0 ± 1.8 mg/L (ANN), respectively. The errors achieved, and best-fitted models were like those for the same bioprocess using offline data and others, which utilized inline spectra for mammalian cell lines as a host.
Assuntos
Análise Espectral Raman , Análise Espectral Raman/métodos , Análise dos Mínimos Quadrados , Glucose/análise , Redes Neurais de Computação , Sobrevivência Celular/efeitos dos fármacos , Ácido Glutâmico/análise , Máquina de Vetores de Suporte , Análise de Componente Principal , Glutamina/análise , Ácido Láctico/análise , Compostos de Amônio/análiseRESUMO
Vaccination against influenza virus can reduce the risk of influenza by 40% to 60%, they rely on the production of neutralizing antibodies specific to influenza hemagglutinin (HA) ignoring the neuraminidase (NA) as an important surface target. Vaccination with standardized NA concentration may offer broader and longer-lasting protection against influenza infection. In this regard, we aimed to compare the potency of a NA displayed on the surface of a VLP with a soluble NA. The baculovirus expression system (BEVS) and the novel virus-free Tnms42 insect cell line were used to express N2 NA on gag-based VLPs. To produce VLP immunogens with high levels of purity and concentration, a two-step chromatography purification process combined with ultracentrifugation was used. In a prime/boost vaccination scheme, mice vaccinated with 1 µg of the N2-VLPs were protected from mortality, while mice receiving the same dose of unadjuvanted NA in soluble form succumbed to the lethal infection. Moreover, NA inhibition assays and NA-ELISAs of pre-boost and pre-challenge sera confirm that the VLP preparation induced higher levels of NA-specific antibodies outperforming the soluble unadjuvanted NA.
Assuntos
Anticorpos Antivirais , Vacinas contra Influenza , Neuraminidase , Infecções por Orthomyxoviridae , Vacinas de Partículas Semelhantes a Vírus , Animais , Neuraminidase/imunologia , Neuraminidase/genética , Vacinas contra Influenza/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Feminino , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Eficácia de Vacinas , Humanos , Vacinação/métodosRESUMO
BACKGROUND/PURPOSE(S): The gut microbiota and its metabolites play crucial roles in pathogenesis of arthritis, highlighting gut microbiota as a promising avenue for modulating autoimmunity. However, the characterization of the gut virome in arthritis patients, including osteoarthritis (OA) and gouty arthritis (GA), requires further investigation. METHODS: We employed virus-like particle (VLP)-based metagenomic sequencing to analyze gut viral community in 20 OA patients, 26 GA patients, and 31 healthy controls, encompassing a total of 77 fecal samples. RESULTS: Our analysis generated 6819 vOTUs, with a considerable proportion of viral genomes differing from existing catalogs. The gut virome in OA and GA patients differed significantly from healthy controls, showing variations in diversity and viral family abundances. We identified 157 OA-associated and 94 GA-associated vOTUs, achieving high accuracy in patient-control discrimination with random forest models. OA-associated viruses were predicted to infect pro-inflammatory bacteria or bacteria associated with immunoglobulin A production, while GA-associated viruses were linked to Bacteroidaceae or Lachnospiraceae phages. Furthermore, several viral functional orthologs displayed significant differences in frequency between OA-enriched and GA-enriched vOTUs, suggesting potential functional roles of these viruses. Additionally, we trained classification models based on gut viral signatures to effectively discriminate OA or GA patients from healthy controls, yielding AUC values up to 0.97, indicating the clinical utility of the gut virome in diagnosing OA or GA. CONCLUSION: Our study highlights distinctive alterations in viral diversity and taxonomy within gut virome of OA and GA patients, offering insights into arthritis etiology and potential treatment and prevention strategies.
Assuntos
Artrite Gotosa , Microbioma Gastrointestinal , Osteoartrite , Viroma , Humanos , Artrite Gotosa/virologia , Artrite Gotosa/microbiologia , Masculino , Osteoartrite/virologia , Osteoartrite/microbiologia , Feminino , Pessoa de Meia-Idade , Estudos de Casos e Controles , Idoso , Metagenômica , Fezes/virologia , Fezes/microbiologiaRESUMO
Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.
Assuntos
Norovirus , Plasmídeos , Rotavirus , Animais , Plasmídeos/genética , Rotavirus/genética , Norovirus/genética , Enterovirus/genética , Células Sf9 , Baculoviridae/genética , Vetores Genéticos/genética , Transfecção/métodos , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Insetos , Linhagem CelularRESUMO
Spike-protein-based pseudotyped viruses were used to evaluate vaccines during the COVID-19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus-like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS-COV-1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild-type VLP pseudotyped virus over 100-fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS-CoV-2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze-thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra- and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high-throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS-CoV-2 based on VLP pseudotyped virus.
RESUMO
African horse sickness is a devastating viral disease of equids. It is transmitted by biting midges of the genus Culicoides with mortalities reaching over 90% in naïve horses. It is endemic to sub-Saharan Africa and is seasonally endemic in many parts of southern Africa. However, outbreaks in Europe and Asia have occurred that caused significant economic issues. There are attenuated vaccines available for control of the virus but concerns regarding the safety and efficacy means that alternatives are sought. One promising alternative is the use of virus-like particles in vaccine preparations, which have the potential to be safer and more efficacious as vaccines against African horse sickness. These particles are best made in a complex, eukaryotic system, but due to technical challenges, this may cause significant economic strain on the developing countries most affected by the disease. Therefore, this review also summarises the success so far, and potential, of recombinant protein expression in plants to reduce the economic strain of production.
RESUMO
Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease worldwide, and a pediatric vaccine is not available. We generated a filamentous RSV-based virus-like particle (VLP) that presents the central conserved region of the attachment protein G. This was achieved by co-expressing the matrix protein, phosphoprotein, nucleoprotein, and a hybrid fusion protein in which the F ectodomain was replaced with the G central region (GCR). The latter is relatively conserved and contains a receptor binding site and hence is a logical vaccine target. The immunogenicity and efficacy of the resulting VLP, termed VLP-GCR, were examined in mice using intranasal application without adjuvant. VLP-GCR induced substantial anti-N antibody levels but very low anti-G antibody levels, even after three vaccinations. In contrast, a VLP presenting prefusion-stabilized fusion (preF) protein instead of GCR induced both high anti-F and anti-nucleoprotein antibody levels, suggesting that our GCR antigen was poorly immunogenic. Challenge of VLP-GCR-vaccinated mice caused increased weight loss and lung pathology, and both VLPs induced mucus in the lungs. Thus, neither VLP is suitable as a vaccine for RSV-naive individuals. However, VLP-preF enhanced the proportion of preF antibodies and could serve as a multi-antigen mucosal booster vaccine in the RSV-experienced population.