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Validation of bioanalytical methods is crucial, especially in the pharmaceutical industry, to determine their suitability for specific purposes and the accuracy of analytical results. The pseudovirion-based neutralization assay (PBNA) is considered the gold standard for detecting and quantifying neutralizing antibodies against human papillomavirus in vaccine development for disease prevention. This paper introduces an improved triple-color PBNA method, capable of simultaneous detection of two or three human papillomavirus (HPV types for use in the development of a 14-valent HPV vaccine candidate. The primary objective was to comprehensively validate the triple-color PBNA method for general vaccine immunogenicity assays. Results show that the method has good specificity, accuracy, precision, linearity, robustness, and applicability. This innovative triple-color PBNA offers an improved approach for large-scale immunogenicity assessment in vaccine development. This study lays a solid foundation that can serve as a guiding paradigm for assessing vaccine responses in preclinical and clinical phases, providing valuable insights to the field.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Testes de Neutralização , Vacinas contra Papillomavirus , Humanos , Testes de Neutralização/métodos , Vacinas contra Papillomavirus/imunologia , Anticorpos Antivirais/sangue , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Vacinas Sintéticas/imunologia , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Imunogenicidade da Vacina , Papillomaviridae/imunologia , Sensibilidade e EspecificidadeRESUMO
A new virus-like particle based vaccine covering 14 types of high-risk and disease-inducing human papillomavirus (HPV) can offer better coverage against HPV-induced diseases, particularly cervical cancers. However, the assessment of immunogenicity of the vaccine is an important task, representing not only its significant clinical characteristics, but also a major challenge, in terms of both the suitability of methods and the clinical sample testing throughput supporting clinical development. This work covers the development and evaluation of a method based on Luminex technology (a coded-bead and flow-cytometric approach) to assess the HPV-type specific total immunoglobulin G (IgG). This method can evaluate the antibodies in sera post immunization against multiple types of HPV simultaneously (i.e., with multiplexing capability), save time and cost, and improve test throughput with higher sensitivity and precision than the classical, plate-based enzyme-linked immunoassay and competitive Luminex-based immunoassays. Using cynomolgus monkeys as model, we demonstrated the good correlation between the results from the pseudovirion-based neutralization assay (PBNA), and the Luminex-based total IgG assay, supporting that the latter method can be considered as a viable, dependable replacement method for the PBNA supporting immunogenicity evaluation of HPV vaccine in preclinical development and clinical investigation.
Assuntos
Alphapapillomavirus , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Animais , Anticorpos Antivirais , Humanos , Imunoensaio/métodos , Imunogenicidade da Vacina , Imunoglobulina G , Macaca fascicularis , Papillomaviridae , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/prevenção & controleRESUMO
Type A and type B influenza viruses (FluA and FluB viruses) are two major human pathogens that share common structural and functional features. FluA and FluB viruses can reassort within each type but never between the types. Here, we bioinformatically analyzed all promoter sequences of FluA and FluB viruses and confirmed the presence of the type-specific promoter elements. We then studied the promoter elements with cell-based in vivo assays and an in vitro replication initiation assay. Our results identified, for the first time, a type-specific promoter element-the nucleotide at position 5 in the 3' end of the viral RNA (vRNA)-that plays a key role(s) in modulating polymerase activity in a type-specific manner. Interestingly, swapping the promoter element between FluA and FluB recombinant viruses showed different tolerances: the replacement of FluA virus-specific U5 with FluB virus-specific C5 in influenza virus A/WSN/33 (H1N1) could be reverted to U5 after 2 to 3 passages, while the replacement of FluB virus-specific C5 with FluA virus-specific U5 in influenza virus B/Yamagata/88 could be maintained, but with significantly reduced replication efficiency. Therefore, our findings indicate that the nucleotide variation at position 5 in the 3' end of the vRNA promoter between FluA and FluB viruses contributes to their RNP incompatibility, which may shed new light on the mechanisms of intertypic exclusion of reassortment between FluA and FluB viruses.IMPORTANCE Genetic reassortment of influenza virus plays a key role in virus evolution and the emergence of pandemic strains. The reassortment occurs extensively within either FluA or FluB viruses but never between them. Here, we bioinformatically compared available promoter sequences of FluA and FluB viruses and confirmed the presence of the type-specific promoter elements. Our in vivo and in vitro mutagenesis studies showed that a type-specific promoter element-the nucleotide at position 5 in the 3' end of vRNA promoters-plays key roles in modulating polymerase activity. Interestingly, FluA and FluB viruses showed different tolerances upon key promoter element swapping in the context of virus infections. We concluded that the nucleotide at position 5 in the 3' end of the vRNA promoters of FluA and FluB viruses is a critical type-specific determinant. This work has implications for further elucidating the mechanisms of the intertypic exclusion of reassortment between FluA and FluB viruses.
Assuntos
Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , RNA Viral/genética , Sequência de Bases , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Proteínas Virais/genética , Replicação ViralRESUMO
Lysine methylation of chromosomal and nuclear proteins is a well-known mechanism of epigenetic regulation, but relatively little is known about the role of this protein modification in signal transduction. Using an RNAi-based functional screening of the SMYD family of lysine methyltransferases (KMTs), we identified SMYD2 as a KMT essential for robust bone morphogenic protein (BMP)- but not TGFß-induced target gene expression in HaCaT keratinocyte cells. A role for SMYD2 in BMP-induced gene expression was confirmed by shRNA knockdown and CRISPR/Cas9-mediated knock-out of SMYD2 We further demonstrate that SMYD2 knockdown or knock-out impairs BMP-induced phosphorylation of the signal-transducing protein SMAD1/5 and SMAD1/5 nuclear localization and interaction with SMAD4. The SMYD2 KMT activity was required to facilitate BMP-mediated signal transduction, as treatment with the SMYD2 inhibitor AZ505 suppressed BMP2-induced SMAD1/5 phosphorylation. Furthermore, we present evidence that SMYD2 likely modulates the BMP response through its function in the cytosol. We show that, although SMYD2 interacted with multiple components in the BMP pathway, it specifically methylated the kinase domain of BMP type II receptor BMPR2. Taken together, our findings suggest that SMYD2 may promote BMP signaling by directly methylating BMPR2, which, in turn, stimulates BMPR2 kinase activity and activation of the BMP pathway.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Transdução de Sinais , Linhagem Celular , Células HEK293 , Humanos , Metilação , Domínios ProteicosRESUMO
Pseudorabies (PR, Aujeszky's disease) is an acute, highly contagious viral disease resulting in major economic losses to the swine industry. PR is endemic in wild and domestic animals, although its natural host is the pig. Here, we report an outbreak of PR in foxes on a fur-producing farm in Yuncheng county, Shandong, China, that were fed pig offal. The diagnosis of PR was based on nervous signs and standard PCR methods and by isolation of PRV from fox brain tissue in Vero cells. The diagnosis was confirmed by an indirect immunofluorescence assay and electron microscopy. Phylogenetic analysis of a partial (804 nt) viral glycoprotein gC gene sequence indicated that it was likely to be a field strain closely related to a cluster of PRV previously identified in China.
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Surtos de Doenças , Raposas/virologia , Pseudorraiva/epidemiologia , Ração Animal , Animais , Composição de Bases , China/epidemiologia , Chlorocebus aethiops , Análise por Conglomerados , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/isolamento & purificação , Herpesvirus Suídeo 1/ultraestrutura , Microscopia Eletrônica de Transmissão , Filogenia , Reação em Cadeia da Polimerase , Pseudorraiva/patologia , Pseudorraiva/virologia , Análise de Sequência de DNA , Suínos , Células Vero , Proteínas do Envelope Viral/genética , Cultura de VírusRESUMO
Lactobacillus helveticus is one of the commonly used starter cultures for manufacturing various fermented dairy products. However, only a few studies have explored the cleavage region preference of L. helveticus with different cell envelope proteinase (CEP) genes. In the present study, we profiled the peptide composition of milk samples fermented by three different L. helveticus strains by means of peptidomics to illustrate their different proteolysis patterns. The result revealed that the differences in peptide profiles of milk samples fermented by different L. helveticus strains were mainly a result of variations in the peptide patterns of the casein fractions, which were correlated with CEP genotypes. This was mainly reflected in the extensiveness of the hydrolysis region of αS1-casein and the degree of ß-casein hydrolysis. Bioactive peptides were mostly derived from the hydrolysis region common to the three L. helveticus strains, and DQHXN-Q32M42 fermentation resulted in the highest diversity and abundance of bioactive peptides and a significant antihypertensive effect in spontaneous hypertension rats.
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With the emergence and rapid spread of new pandemic variants, especially variants of concern (VOCs), the development of next-generation vaccines with broad-spectrum neutralizing activities is of great importance. In this study, SCTV01C, a clinical stage bivalent vaccine based on trimeric spike extracellular domain (S-ECD) of SARS-CoV-2 variants Alpha (B.1.1.7) and Beta (B.1.351) with a squalene-based oil-in-water adjuvant was evaluated in comparison to its two corresponding (Alpha and Beta) monovalent vaccines in mouse immunogenicity studies. The two monovalent vaccines induced potent neutralizing antibody responses against the antigen-matched variants, but drastic reductions in neutralizing antibody titers against antigen-mismatched variants were observed. In comparison, the bivalent vaccine SCTV01C induced relatively higher and broad-spectrum cross-neutralizing activities against various SARS-CoV-2 variants, including the D614G variant, VOCs (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529), variants of interest (VOIs) (C.37, B.1.621), variants under monitoring (VUMs) (B.1.526, B.1.617.1, B.1.429, C.36.3) and other variants (B.1.618, 20I/484Q). All three vaccines elicited potent Th1-biased T-cell immune responses. These results provide direct evidence that variant-based multivalent vaccines could play important roles in addressing the critical issue of reduced protective efficacy against the existing and emerging SARS-CoV-2 variants.
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Hepatitis C virus(HCV)infection is a global public health problem, primarily triggering acute and chronic liver hepatitis. Owing to the narrow host range, a suitable animal model is still lacking, hindering study of viral pathogenesis, immune control,and prophylactic vaccine development. There has no relevant evidence that homologs of HCV originating from the animal may have the potential to cross the species barrier to cause human disease until recently. Several agents discovered that new viruses related to HCV, including HCV and GBV-like viruses(belonging to the Hepacivirus and Pegivirus genera, respectively), in small wild mammals (e.g., rodents and bats)and domesticated animals(e.g., dogs, horses, and cattle). Genetic and biological characterization of these novel HCV-related viruses may provide novel insight into the origins, pathogenesis, and immune response to HCV infection in humans. In this review, we introduce the gene characteristics of HCV, concerned viruses, and many newly discovered viruses closely related to HCV-like viruses. The exploration of their natural reservoirs will be performed, and we then discuss possible theories regarding the origin of this important viral human pathogen.
Assuntos
Reservatórios de Doenças/virologia , Hepacivirus/fisiologia , Hepatite C/virologia , Animais , Bovinos , Cães , Hepacivirus/classificação , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Cavalos , HumanosRESUMO
Androgen receptor (AR) antagonist MDV3100 is the first therapeutic approach in treating castration-resistant prostate cancer (CRPC), but tumours frequently become drug resistant via multiple mechanisms including AR amplification and mutation. Here we identify the small molecule Ailanthone (AIL) as a potent inhibitor of both full-length AR (AR-FL) and constitutively active truncated AR splice variants (AR-Vs). AIL binds to the co-chaperone protein p23 and prevents AR's interaction with HSP90, thus resulting in the disruption of the AR-chaperone complex followed by ubiquitin/proteasome-mediated degradation of AR as well as other p23 clients including AKT and Cdk4, and downregulates AR and its target genes in PCa cell lines and orthotopic animal tumours. In addition, AIL blocks tumour growth and metastasis of CRPC. Finally, AIL possesses favourable drug-like properties such as good bioavailability, high solubility, lack of CYP inhibition and low hepatotoxicity. In general, AIL is a potential candidate for the treatment of CRPC.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Terapia de Alvo Molecular , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Quassinas/uso terapêutico , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Chaperonas Moleculares , Metástase Neoplásica , Nitrilas , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Fosfoproteínas , Prostaglandina-E Sintases , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Quassinas/administração & dosagem , Quassinas/farmacocinética , Quassinas/farmacologia , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transcription has been linked to DNA damage. How the most highly transcribed mammalian ribosomal (rDNA) genes maintain genome integrity in the absence of transcription-coupled DNA damage repair is poorly understood. Here, we report that ABH2/ALKBH2, a DNA alkylation repair enzyme, is highly enriched in the nucleolus. ABH2 interacts with DNA repair proteins Ku70 and Ku80 as well as nucleolar proteins nucleolin, nucleophosmin 1, and upstream binding factor (UBF). ABH2 associates with and promotes rDNA transcription through its DNA repair activity. ABH2 knockdown impairs rDNA transcription and leads to increased single-stranded and double-stranded DNA breaks that are more pronounced in the rDNA genes, whereas ABH2 overexpression protects cells from methyl-methanesulfonate-induced DNA damage and inhibition of rDNA transcription. In response to massive alkylation damage, ABH2 rapidly redistributes from the nucleolus to nucleoplasm. Our study thus reveals a critical role of ABH2 in maintaining rDNA gene integrity and transcription and provides insight into the ABH2 DNA repair function.