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An imaged capillary isoelectric focusing (icIEF)-based method was developed and validated as a multi-attribute method for a bispecific antibody (BsAb). First, as the traditional application of the icIEF method, it serves as an identity assay and purity assay for the BsAb. Second, the method can also be used as an impurity assay for the homodimer monoclonal antibodies generated during BsAb assembly. The homodimer impurity analysis for BsAb is usually done by hydrophobic interaction chromatography methods in the industry. The icIEF method has good sensitivity (down to 4 µg/mL in a limit of quantitation) when UV fluorescence detection is used, which detects the native fluorescence of proteins. This is the first report that an icIEF method has been applied as impurity assay.
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The size variant, which can be measured by capillary electrophoresis sodium dodecyl sulfate (CE-SDS), is a critical quality attribute of monoclonal antibodies (mAbs). The CE-SDS size heterogeneity can hardly be identified by tandem mass spectrometry, which is an intractable obstacle of mAb development and quality control across the industry. We analyzed the purity of an anti-vascular endothelial growth factor receptor 2 (VEGFR-2) mAb, an antagonist of the human VEGFR-2, through reduced CE-SDS and observed glycosylated heavy chain heterogeneity. The heterogeneity has potential impact on safety, efficacy, and stability of drugs for clinical use. Therefore, it should be characterized so as to evaluate its potential risk. In order to identify the heterogeneity, we used mass spectrometry to confirm that the molecular size heterogeneity was not due to peptide bond cleavage in the heavy chain. Subsequently, we employed mass-spectrometry-glycosylation profiling and CE-SDS analysis of various glycosidase-treated samples, in addition to the preparation of mAb references with different glycoforms. Ultimately, we demonstrated that the heavy chain heterogeneity was induced by different levels of galactosylation modifications which will potentially impact the efficacy of antibody drugs (i.e., complement-dependent cytotoxicity). In this study, potential risk caused by heavy chain size heterogeneity was evaluated, which addressed the obstacle of mAb development and quality control. Therefore, this study offers a feasible approach for the investigation and identification of heavy chain heterogeneity in reduced CE-SDS, providing a novel strategy for mAb quality control and evaluation.
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Anticuerpos Monoclonales , Electroforesis Capilar , Espectrometría de Masas , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Electroforesis Capilar/métodos , Glicosilación , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Humanos , Cadenas Pesadas de Inmunoglobulina/análisis , Cadenas Pesadas de Inmunoglobulina/química , Dodecil Sulfato de Sodio/química , Animales , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Imaged capillary isoelectric focusing (icIEF) and ion-exchange chromatography (IEX) are two essential techniques that are routinely used for charge variant analysis of therapeutic monoclonal antibodies (mAbs) during their development and in quality control. These two techniques that separate mAb charge variants based on different mechanisms and IEX have been developed as front-end separation techniques for online mass spectrometry (MS) detection, which is robust for intact protein identification. Recently, an innovative, coupled icIEF-MS technology has been constructed for protein charge variant analysis in our laboratory. In this study, icIEF-MS developed and strong cation exchange (SCX)-MS were optimized for charge heterogeneity characterization of a diverse of mAbs and their results were compared based on methodological validation. It was found that icIEF-MS outperformed SCX-MS in this study by demonstrating outstanding sensitivity, low carryover effect, accurate protein identification, and higher separation resolution although SCX-MS contributed to higher analysis throughput. Ultimately, integrating our novel icIEF-HRMS analysis with the more common SCX-MS can provide a promising and comprehensive strategy for accelerating the development of complex protein therapeutics.
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Anticuerpos Monoclonales , Focalización Isoeléctrica Capilar , Anticuerpos Monoclonales/química , Espectrometría de Masas/métodos , Focalización Isoeléctrica/métodos , Cromatografía por Intercambio Iónico/métodosRESUMEN
Because the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is the immunodominant antigen, the S protein and its receptor-binding domain (RBD) are both targets currently to be genetically engineered for designing the broad-spectrum vaccine. In theory, the expressed protein exists as a set of variants that are roughly the same but slightly different, which depends on the protein expression system. The variants can be phenotypically manifested as charge heterogeneity. Here, we attempted to depict the charge heterogeneity of the trimeric SARS-CoV-2 RBD by using capillary isoelectric focusing with whole-column imaging detection (cIEF-WCID). In its nature form, the electropherogram fingerprints of the trimeric RBD were presented under optimized experimental conditions. The peaks of matrix buffers can be fully distinguishable from peaks of trimeric RBD. The isoelectric point (pI) was determined to be within a range of 6.67-9.54 covering the theoretical pI of 9.02. The fingerprints of three batches of trimeric RBDs are completely the same, with the intra-batch and batch-to-batch relative standard deviations (RSDs) of both pI values and area percentage of each peak no more than 1.0%, indicating that the production process is stable and this method can be used to surveillance the batch-to-batch consistency. The fingerprint remained unchanged after incubating at 37 °C for 7 d and oxidizing by 0.015% H2O2. In addition, the fingerprint was destroyed when adjusting the pH value to higher than 10.0 but still stable when the pH was lower than 4.0. In summary, the cIEF-WCID fingerprint can be used for the identification, batch-to-batch consistency evaluation, and stability study of the trimeric SARS-CoV-2 RBD, as part of a quality control strategy during the potential vaccine production.
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COVID-19 , SARS-CoV-2 , Humanos , Focalización Isoeléctrica/métodos , Focalización Isoeléctrica Capilar , Peróxido de Hidrógeno , Unión ProteicaRESUMEN
Rabies is a viral disease that is nearly 100% fatal once clinical signs and symptoms develop. Post-exposure prophylaxis can efficiently prevent rabies, and antibody (Ab) induction by vaccination or passive immunization of human rabies immunoglobulin (HRIG) or monoclonal antibodies (mAbs) play an integral role in prevention against rabies. In addition to their capacity to neutralize viruses, antibodies exert their antiviral effects by antibody-dependent cellular cytotoxicity (ADCC), which plays an important role in antiviral immunity and clearance of viral infections. For antibodies against rabies virus (RABV), evaluation of ADCC activity was neglected. Here, we developed a robust cell-based reporter gene assay (RGA) for the determination of the ADCC activity of anti-RABV antibodies using CVS-N2c-293 cells, which stably express the glycoprotein (G) of RABV strain CVS-N2c as target cells, and Jurkat cells, which stably express FcγR⠢a and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT-luc/FcγR⠢a cells). The experimental parameters were carefully optimized, and the established ADCC assay was systematically validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 guideline. We also evaluated the ADCC activity of anti-RABV antibodies, including mAbs, HRIG, and vaccine induced antisera, and found that all test antibodies exhibited ADCC activity with varied strengths. The established RGA provides a novel method for evaluating the ADCC of anti-RABV antibodies.
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Vacunas Antirrábicas , Rabia , Humanos , Anticuerpos Antivirales , Genes Reporteros , Vacunas Antirrábicas/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Anticuerpos Monoclonales , Glicoproteínas/genética , AntiviralesRESUMEN
Norovirus-like particle (VLP) vaccine is promising against human norovirus infection. Unfortunately, genetic diversity of norovirus hindered the development of this vaccine. In this study, the immunogenicity of norovirus VLPs induced by the endemic GII.4 and the epidemic GII.17 genotypes, and the cross-reactivity between them as well as GI.1 and GII.3 VLPs were evaluated in mice by using serum IgG and histo-blood group antigen (HBGA) blocking antibodies as index. Results showed well immunogenicity of both GII.4 and GII.17 VLPs in mice. Serum IgG GMT (Geometric Mean Titer) were 3.63 (GII.4) and 3.88 (GII.17) respectively, and sustained to the 15th week. The HBGA blocking antibodies were 130 (GII.4) and 360 (GII.17) respectively at the end of the 4th week. Additionally, there was a dramatically statistical difference found in the cross-reactivity within genogroup (GII.3, GII.4 and GII.17) (p < .001), and also showed similar difference between genogroups (GI.1 vs. GII.3, GII.4 and GII.17) (p < .001). Summarized the pPICZa pichi pichia expression system showed a potential to be the alternative for expression of norovirus VLPs in secretion form, and the little cross-reactivity found between the endemic strain and the epidemic strain provides an evident for the consideration of selecting candidates of norovirus vaccine strains.
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Anticuerpos Antivirales/sangre , Infecciones por Caliciviridae/inmunología , Reacciones Cruzadas/inmunología , Gastroenteritis/virología , Variación Genética/inmunología , Genotipo , Norovirus/genética , Norovirus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Reacciones Cruzadas/genética , Enfermedades Endémicas , Humanos , Ratones , Ratones Endogámicos BALB C , Organismos Libres de Patógenos Específicos , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Vacunas Virales/normasRESUMEN
In 2010, Rotarix was found to be contaminated with infectious porcine circovirus type 1 (PCV1). In China, the Lanzhou lamb rotavirus (LLR) vaccine is the only vaccine used to prevent rotavirus disease. From 2006 to September 2014, more than 54 million doses of LLR vaccines have been lot released. It is a safety issue whether PCV1 is present in the LLR vaccine. Although the cell substrate of LLR, bovine kidney (BK), is different from that of Rotarix, we have investigated the cell's permissivity for PCV1 by both infectivity and full-length PCR analysis. We have assessed the LLR using a quantitative PCR (qPCR) assay. A total of 171 random batches of LLR final products over a period of 5 years were tested, and no PCV1 was detected (0/171). Infectivity studies showed that two strains of PCV1, the PCV1-prototype, which was derived from PK-15 cells, and the mutant, PCV1-GSK, which was isolated from Rotarix, were capable of replicating in BK cells over a wide m.o.i. ranging from 10 to 0.01. After culture for 6 days, copies of PCV1-prototype DNA were higher than those of PCV1-GSK on average. The genome of the virus was detected at 6 days post-infection. In summary, the LLR vaccine is free of PCV1. Nevertheless, because PCV1 can replicate in the BK cell substrate, manufacturers need to be vigilant in monitoring for this adventitious agent.
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Circovirus/crecimiento & desarrollo , ADN Viral/genética , Contaminación de Medicamentos/prevención & control , Células Epiteliales/virología , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/análisis , Animales , Bovinos , Línea Celular , China , Circovirus/genética , Circovirus/aislamiento & purificación , ADN Viral/aislamiento & purificación , Células Epiteliales/citología , Riñón/citología , Riñón/virología , Control de Calidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Rotavirus/inmunología , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/virología , Vacunas contra Rotavirus/biosíntesis , Oveja Doméstica , Porcinos , Vacunas Atenuadas/análisis , Vacunas Atenuadas/biosíntesisRESUMEN
SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.
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Vacunas contra la COVID-19 , COVID-19 , Animales , COVID-19/prevención & control , SARS-CoV-2/genética , Espectrometría de Masas en Tándem , Saccharomyces cerevisiae , Vacunas Sintéticas , MamíferosRESUMEN
The development and optimization of Antibody-Drug Conjugates (ADCs) hinge on enhanced analytical and bioanalytical characterization, particularly in assessing critical quality attributes (CQAs). The ADC's potency is largely determined by the average number of drugs attached to the monoclonal antibody (mAb), known as the drug-to-antibody ratio (DAR). Furthermore, the drug load distribution (DLD) influences the therapeutic window of the ADC, defining the range of dosages effective in treating diseases without causing toxic effects. Among CQAs, DAR and DLD are vital; their control is essential for ensuring manufacturing consistency and product quality. Typically, hydrophobic interaction chromatography (HIC) or reversed-phase liquid chromatography (RPLC) with UV detector have been used to quantitate DAR and DLD in quality control (QC) environment. Recently, Native size-exclusion chromatography-mass spectrometry (nSEC-MS) proves the potential as a platformable quantitative method for characterizing DAR and DLD across various cysteine-linked ADCs in research or early preclinical development. In this work, we established and assessed a streamlined nSEC-MS workflow with a benchtop LC-MS platform, to quantitatively monitor DAR and DLD of different chemotype and drug load level cysteine-linked ADCs. Moreover, to deploy this workflow in QC environment, complete method validation was conducted in three independent laboratories, adhering to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. The results met the predefined analytical target profile (ATP) and performance criteria, encompassing specificity/selectivity, accuracy, precision, linearity, range, quantification/detection limit, and robustness. Finally, the method validation design offers a reference for other nSEC-MS methods that are potentially used to determine the DAR and DLD on cysteine-linker ADCs. To the best of our knowledge, this study is the first reported systematic validation of the nSEC-MS method for detecting DAR and DLD. The results indicated that the co-validated nSEC-MS workflow is suitable for DAR and DLD routine analysis in ADC quality control, release, and stability testing.
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Cromatografía en Gel , Cisteína , Inmunoconjugados , Espectrometría de Masas , Inmunoconjugados/química , Inmunoconjugados/análisis , Cisteína/química , Reproducibilidad de los Resultados , Cromatografía en Gel/métodos , Espectrometría de Masas/métodos , Modelos Lineales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Límite de Detección , Humanos , Flujo de TrabajoRESUMEN
Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.
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Brentuximab Vedotina , Cisteína , Inmunoconjugados , Brentuximab Vedotina/química , Brentuximab Vedotina/análisis , Cisteína/química , Cisteína/análisis , Inmunoconjugados/química , Inmunoconjugados/análisis , Cromatografía Líquida con Espectrometría de Masas , Mapeo Peptídico/métodosRESUMEN
Anti-drug antibody (ADA) positivity is correlated with disease relapse risk when treated with monoclonal antibody (mAb) therapeutics. ADA evaluation can assist with interpreting pharmacokinetic, pharmacological, and toxicology results. Here, we established an ADA assay based on two steps of acid dissociation combined with a bridging immunoassay to provide a comprehensive validation strategy. The three-tiered sample analysis process included screening, confirmation, and titration assays using therapeutic HLX26 (targeting lymphocyte activation gene-3 [LAG-3]) as an example. The cut points were determined by testing 50 individual normal human serum samples, including screening cut point (SCP) (SNR: 1.08), confirmatory cut point (CCP) (% inhibition: 12.65), and titration cut point (TCP) (sample-to-noise ratio [SNR]: 1.17). The assay sensitivity, low positive control (LPC), and high positive control (HPC) titer acceptable range were also set up as 33.0 ng/mL, 41.0 ng/mL, and 320-1280, respectively. After full validation, both the intra-assay and inter-assay precision testing passed with coefficient of variations (CVs) < 20%. The assay enabled excellent drug tolerance up to 768.0 µg/mL at the HPC level and 291.0 µg/mL at the LPC level, while the tolerance of target interference was up to 74.0 ng/mL of soluble LAG3. Moreover, no false-positive results were observed in the presence of 5% hemolyzed serum samples and 150 mg/dL of triglyceride in the serum samples, no hook effect was observed, and the stability performed normally under room temperature for 24 h, 2-8 °C for 7 d, and six freeze/thaw cycles. In summary, this ADA assay is feasible and could be used for evaluating the immunogenicity of HLX26 in clinical trials.
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Objective: This study aimed to update the genetic diversity of Rotavirus (RV) infections in children under five years old in Beijing, China. Methods: A 5-year active hospital-based surveillance for sporadic acute gastroenteritis (AGE) from January 2018 to December 2022 in the capital of China was performed. A total of 748 fecal samples from AGE patients were collected for followed by RV antigen detection by ELSIA, RNA detection by reverse transcription PCR, G/P genotyping and phylogenetic analyzing. Results: RV antigen was detected in 11.0% of the collected samples, with 54 samples confirmed to be RV RNA positive. G9 and G8 genotypes were identified in 43 (79.6%) and 7 (13.0%) samples, respectively, all of which were allocated to P[8]. The predominant G/P combination was G9P[8] (79.6%), following by G8P[8] (13.0%), G4P[8] (5.6%) and G3P[8] (1.9%). A significant change in G/P-type distribution was observed, with the G9P[8] being predominant from 2018 to 2021, followed by the emergence of an uncommon G8P[8] genotype, which was first reported in 2021 and became predominant in 2022. Blast analysis showed that one G1 isolate had a high similarity of 99.66% on nucleotide acid with RotaTeq vaccine strain with only one amino acid difference L150V. Additionally, one P[8] isolate was clustered into a branch together with RotaTeq vaccine strain G6P[8]. Conclusions: The study reveals that G8P[8] has become the predominant genotype in pediatric outpatients in China for the first time, indicating a significant change in the composition of RV genetic diversity. The importance of RVA genotyping in surveillance is emphasized, as it provides the basis for new vaccine application and future vaccine efficacy evaluation.
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Anti-CD25 antibodies have been approved for renal transplantation and has been used prior to and during transplantation by the Food and Drug Administration (FDA). However, no reported bioassays have been reflected the mechanism of action (MOA) of anti-CD25 antibodies. Here, we describe the development and validation of a reporter gene assay (RGA) based on the engineered C8166-STAT5RE-Luc cells expressing endogenous IL-2 receptors and a STAT5-inducible element-driven firefly luciferase in C8166 cell lines. The RGA was fully validated according to the International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for the Human Use-Q2 (ICH-Q2). After optimization, the assay showed excellent specificity, linearity, accuracy, precision, and robustness. Due to the MOA relatedness and the excellent assay performance, the RGA is suitable for exploring the critical quality attributes (CQAs), release inspection, comparability and stability of anti-CD25 mAbs.
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Introduction: Human sapovirus (HuSaV) is an enteric virus responsible for sporadic cases and outbreaks of acute gastroenteritis (AGE) globally. A seven-year active surveillance study was conducted to investigate the molecular epidemiology of HuSaVs associated with AGE outbreaks in Chaoyang District of Beijing Municipality, China from January 2015 to December 2021. Methods: Fecal and anal swab samples were obtained from patients experiencing AGE outbreaks. HuSaVs were identified through reverse transcription polymerase chain reaction (RT-PCR), and partial viral protein 1 (VP1) sequences (approximately 434 base pairs) were utilized for genotyping, single nucleotide polymorphism (SNP) analysis, and phylogenetic examination. Results: HuSaVs were identified in 71 AGE outbreaks, demonstrating a detection rate of 10.5%, second only to norovirus. The primary demographic affected by HuSaV were children under the age of 5 in kindergarten settings. Infection rates tended to peak during two distinct periods: May to June and September to December. Upon genotyping, seven distinct genotypes emerged. GII.3 was the most prevalent, accounting for 54.9% of cases, followed by GI.1 (12.7%), GI.2 (9.9%), GII.5 (7.0%), GI.5 (2.8%), GI.6 (1.4%), GII.1 (1.4%), and untyped cases (9.9%). A phylogenetic analysis of GII.3 identified three distinct groups, with 15 notable SNPs observed. Conclusions: This study offers a comprehensive analysis of the persistent prevalence of HuSaV outbreaks in Chaoyang District, Beijing Municipality, China. Over time, the diversity of HuSaV subtypes has shifted, and it is now recognized as the second leading viral agent responsible for AGE outbreaks. This highlights the importance of ongoing surveillance in the future.
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Owing to the limitation of in vitro culture of human noroviruses (HuNoVs), the development of HuNoV vaccines has to depend on the self-assembling virus-like particles (VLPs) with capsid protein expression. The heterogeneity of artificial VLPs exert an impact on the immunogenicity, and should be considered as one of the factors in vaccine evaluation. In this study, we biochemically finger print the HuNoV VLPs with different genogroups, genotypes and sub-genotypes which constitute for a candidate vaccine, by using capillary isoelectric focusing with whole column imaging detection (cIEF-WCID). The electropherograms of GI.1, GII.3, GII.4 and GII.17 VLPs in fluorescence signal were described in the monomer VP1 forms after degenerated by 8 M urea. The four HuNoV VLPs showed different properties in electropherogram finger prints. The finger prints were also reproducible within a certain concentration range (approx. 150 â¼ 20 ug/ml). This method can also tell the changes of pI finger-print patterns when the expired HoNoV VLPs were tested. In conclusion, cIEF-WCID shows great promise for evaluating the production consistency of HuNoV VLP vaccine.
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Infecciones por Caliciviridae , Norovirus , Cápside , Proteínas de la Cápside/química , Genotipo , Humanos , Focalización Isoeléctrica/métodosRESUMEN
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Células CHO , COVID-19/prevención & control , COVID-19/virología , Cromatografía Líquida de Alta Presión/métodos , Dicroismo Circular , Células Clonales , Cricetinae , Cricetulus , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Punto Isoeléctrico , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
A randomized, double-blind, placebo-controlled multicenter trial was conducted in healthy Chinese infants to assess the efficacy and safety of a hexavalent live human-bovine reassortant rotavirus vaccine (HRV) against rotavirus gastroenteritis (RVGE). A total of 6400 participants aged 6-12 weeks were enrolled and randomly assigned to either HRV (n â= â3200) or placebo (n â= â3200) group. All the subjects received three oral doses of vaccine four weeks apart. The vaccine efficacy (VE) against RVGE caused by rotavirus serotypes contained in HRV was evaluated from 14 days after three doses of administration up until the end of the second rotavirus season. VE against severe RVGE, VE against RVGE hospitalization caused by serotypes contained in HRV, and VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were also investigated. All adverse events (AEs) were collected for 30 days after each dose. Serious AEs (SAEs) and intussusception cases were collected during the entire study. Our data showed that VE against RVGE caused by serotypes contained in HRV was 69.21% (95%CI: 53.31-79.69). VE against severe RVGE and RVGE hospitalization caused by serotypes contained in HRV were 91.36% (95%CI: 78.45-96.53) and 89.21% (95%CI: 64.51-96.72) respectively. VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were 62.88% (95%CI: 49.11-72.92), 85.51% (95%CI: 72.74-92.30) and 83.68% (95%CI: 61.34-93.11). Incidences of AEs from the first dose to one month post the third dose in HRV and placebo groups were comparable. There was no significant difference in incidences of SAEs in HRV and placebo groups. This study shows that this hexavalent reassortant rotavirus vaccine is an effective, well-tolerated, and safe vaccine for Chinese infants.
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Infecciones por Enterovirus , Gastroenteritis , Infecciones por Rotavirus , Vacunas contra Rotavirus , Rotavirus , Administración Oral , Animales , Bovinos , China , Gastroenteritis/epidemiología , Humanos , Lactante , Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/efectos adversos , Vacunación , Vacunas Atenuadas , Vacunas CombinadasRESUMEN
BACKGROUND: A randomized, double-blind, placebo-controlled multicenter trial was conducted in healthy Chinese infants to assess the efficacy, immunogenicity and safety of a novel trivalent live human-lamb reassortant rotavirus vaccine (LLR3) against rotavirus gastroenteritis (RVGE). METHODS: Healthy children aged 6-13 weeks were enrolled and randomized (1:1) to either 3 oral doses of LLR3 or placebo according to a 0, 1, 2 month schedule. The objectives were to evaluate vaccine efficacy (VE) against RVGE of any-severity, severe RVGE (sRVGE) and inpatient caused by rotavirus serotypes contained in the vaccine and not contained in the vaccine after the third dose. Immunogenicity was also assayed in a subgroup. All adverse events (AEs) were collected from 30 min after each dose for immediate reaction, even to the entire study period, including the serious AEs (SAEs) and intussusception. RESULTS: VE against RVGE of any-severity, sRVGE and inpatient caused by any serotype was 56.6% (95% CI: 50.7, 61.8), 70.3% (95% CI: 60.6, 77.6) and 74.0% (95% CI: 57.5, 84.1) respectively. VE against RVGE of any-severity, sRVGE caused by serotypes not contained in vaccine were 54.2% (95% CI: 47.5, 60.1) and 70.4% (95% CI: 60.4, 77.9). The rate of seroconversion and four-fold increase of rotavirus serotype G2-, G3-, and G4-specific IgA is 60.8%, 58.0%, and 60.6% in vaccine group, which was higher than 21.35%, 22.7%, and 23.1% in placebo group (p < 0.0001 for G2, G3, G4), as well as the Geometric Mean Titer (GMT). Through the entire trial, 65.91% and 67.79% of participants reported at least one AE, and 0.02% and 0.02% reported SAEs in the vaccine and placebo groups, respectively. Two intussusception cases were reported both in vaccine and placebo group. CONCLUSIONS: In Chinese infants, LLR3 provided a substantial protection against RVGE of any-severity, sRVGE and inpatient caused by any serotype, and showed well immunogenicity and safety.
Asunto(s)
Gastroenteritis , Infecciones por Rotavirus , Vacunas contra Rotavirus , Rotavirus , Adolescente , Animales , Anticuerpos Antivirales , Niño , China , Método Doble Ciego , Gastroenteritis/prevención & control , Humanos , Lactante , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/efectos adversos , Ovinos , Vacunas Atenuadas/efectos adversosRESUMEN
In this study, the complete genomic sequence of a rabies virus isolate HN10, recovered from brain tissue of a rabid patient in China, was determined. This is the first Chinese street isolate that has been fully characterized. The overall organization of this virus is typical of that observed for all other rabies viruses. Alignments of amino acid sequences of the phosphoprotein, glycoprotein and large protein of HN10 with those of other rabies viruses were used to examine the extent of conservation of known functional regions. Phylogenetic analysis using either the complete or partial genomic sequence of HN10 determined that this isolate is most closely associated with viruses previously shown to circulate in Guangxi and Hunan provinces. In addition, of all vaccine strains used for comparison, the attenuated Chinese vaccine strain CTN181 is most closely related to HN10.
Asunto(s)
Genoma Viral , Virus de la Rabia/genética , Rabia/virología , Secuencia de Aminoácidos , Secuencia de Bases , Encéfalo/virología , China , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Filogenia , ARN Viral/análisis , ARN Viral/genética , Virus de la Rabia/aislamiento & purificación , Alineación de Secuencia , Proteínas Virales/análisisRESUMEN
Human rabies virus vaccine strain CTN181 from China was sequenced. The overall length of the genome was 11,923 nucleotides (nt), comprising a leader sequence of 58 nt, nucleoprotein (N) gene of 1353 nt, phosphoprotein (P) gene of 894 nt, matrix protein (M) gene of 609 nt, glycoprotein (G) gene of 1575 nt, RNA-dependent RNA polymerase (RdRp, L) gene of 6387 nt, and a trailer region of 70 nt. The five monocistrons are separated by intergenic regions (IGRs) of 2, 5, 5 and 24 nucleotides (nt), respectively. Two obvious differences between CTN181 and the other rabies virus vaccine strains were (1) the putative stop/polyadenylation signal of the G gene has only one poly (A) tract for CTN181, and (2) the start of the open reading frame for L has two repeats of ATG for CTN181. Both were similar to the SHBRV-18 (silver-haired bat-associated RV strain 18) strain. In addition, some mutations and new functional regions were discovered that are presumed crucial to the function of leader region and L protein. There is an equal role for all five genes in the phylogenetics of rabies virus.