A native SEC-MS workflow and validation for analyzing drug-to-antibody ratio and drug load distribution in cysteine-linked antibody-drug conjugates.

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.

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity.

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.

Using capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and liquid chromatograph mass spectrometry (LC-MS) to identify glycosylated heavy chain heterogeneity in the anti-VEGFR-2 monoclonal antibody.

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.

Human rotavirus strains circulating among children in the capital of China (2018-2022)_ predominance of G9P[8] and emergence ofG8P[8].

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.

Genotypes Diversity of Acute Gastroenteritis Outbreaks Caused by Human Sapovirus - Beijing Municipality, China, 2015-2021.

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.

Establishing a novel and sensitive assay for bioactivity determination of anti-CD25 antibodies.

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.

Method validation of a bridging immunoassay in combination with acid-dissociation and bead treatment for detection of anti-drug antibody.

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.

Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques.

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.

Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection.

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.

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti-rabies virus glycoprotein antibodies.

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 FcγRⅢa and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT-luc/FcγRⅢa 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.

Efficacy, safety and immunogenicity of hexavalent rotavirus vaccine in Chinese infants.

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€‹= â€‹3200) or placebo (n â€‹= â€‹3200) 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.

Finger printing human norovirus-like particles by capillary isoelectric focusing with whole column imaging detection.

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 ∼ 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.

Quality comparability assessment of a SARS-CoV-2-neutralizing antibody across transient, mini-pool-derived and single-clone CHO cells.

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.

The endemic GII.4 norovirus-like-particle induced-antibody lacks of cross-reactivity against the epidemic GII.17 strain.

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.

Efficacy, immunogenicity and safety of a trivalent live human-lamb reassortant rotavirus vaccine (LLR3) in healthy Chinese infants: A randomized, double-blind, placebo-controlled trial.

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.

Association of human leukocyte antigen alleles and supertypes with immunogenicity of oral rotavirus vaccine given to infants in China.

Rotavirus (RV) vaccines show distinct immunogenicity in dozens of clinical trials, which is associated with multiple host and environmental factors. Previous research has demonstrated that the highly polymorphic human leukocyte antigen (HLA) system plays an essential role in regulating immune response to a variety of vaccines. This study aims to investigate the relationship between HLA polymorphisms and immunogenicity of RV vaccine.A nested case-control study was carried out among infants enrolled in phase III clinical trial of trivalent human-lamb reassortant vaccine (RV3) in Henan province, China. Serum RV specific immunoglobulin A (RV-IgA) was detected before and after a 3-dose vaccination series, followed by calculation of seroconversion rates. Seroconversion was defined as a 4-fold or greater increase in RV-IgA titers between pre-vaccination and 1-month post-dose 3 vaccination. The infants who seroconverted were defined as responders, and the others without seroconversion were considered as non-responders. Their HLA genotypes were obtained by using the sequence-based typing method. The HLA allele and supertype frequencies of 2 groups were analyzed statistically.Eighty-three of 133 infants seroconverted after vaccination. Twenty-one HLA-A, 45 HLA-B, 24 HLA-Cw, 29 HLA-DRB1 and 16 HLA-DQB1 distinct alleles were detected. The frequency of HLA-B4001 (corrected P = .01, adjusted OR = 0.152, 95% CI = 0.048-0.475) in non-responder group was significantly higher than that in responder group. Furthermore, significant association was found between HLA-B44 supertype (corrected P = .02, adjusted OR = 0.414, 95% CI = 0.225-0.763) and RV non-response.Certain HLA allele (HLA-B4001) and supertype (HLA-B44) are potentially associated with non-response after immunization with the novel RV3 vaccine in Chinese infants.

Porcine circovirus type 1 was undetected in vaccine but could be cultured in the cell substrate of Lanzhou lamb rotavirus vaccine.

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.

Detailed analysis of BALB/c mice challenged with wild type rotavirus EDIM provide an alternative for infection model of rotavirus.

Mouse is one of the infection animal models for rotavirus. Since the optimal age of mouse sensitive to rotavirus infection thus far has not been unified, we elucidated clinical symptoms, immune responses and pathological changes of mice in different ages after challenged by murine rotavirus wild strain EDIM (Epidemic Diarrhea of Infant Mice) to provide data for the estimation. One-week-old, two-week-old, and three-week-old BALB/c mice were inoculated with EDIM in the challenge dose of 235 ID50, 470 ID50 and 705 ID50 respectively and were compared to mock-infected controls. Diarrhea illness, mobility, bodyweight were recorded, viral shedding and immune responses including serum IgA, fecal sIgA were detected, and small intestine tissue was evaluated for virus distribution and pathological changes. All the mice in one-week-old and two-week-old groups were completely unavoidable to be infected by EDIM and have been found to be malaise, activity reduced and even diarrhea, while three-week-old mice partly resist the challenge with 40% mice free from diarrhea. Meanwhile, EDIM infection has greater impact to the bodyweight of two-week-old group than those of one-week-old, three-week-old (0.9860 vs 1.2340, 1.2375g/day). One peak of virus shedding in three groups was observed in day 1-2 post infection, but the duration shortened with age increase. Feces sIgA in both two-week-old and three-week-old groups began to increase in day 4, 2-3days earlier than that in one-week-old group, and grow to the peak in day 8, which is about 2 fold of that in one-week-old group. Stronger serum IgA response was found in two-week-old group, it increased to the peak in day 15 and the level was 2 fold of three-week-old group and 4 fold of one-week-old group. The pathological changes included vacuolar degeneration, edema and congestion of intestinal wall, integrity destruction of enteric epithelium, and the changes relieved with the increase of age. Besides, rotavirus particles were found in small intestine tissues, especially in the surface and crypt of villi. In conclusion, the two-week-old mice were more sensitive to EDIM infection and initiated more effective immune response. In combination with that 14days old mice equals to 2 months infant when the first dose of rotavirus vaccine should be administrated, two-week-old mice is preferred to be used as infection model for the study of pathogenicity and immunogenicity of rotavirus.

Evaluation of oral Lanzhou lamb rotavirus vaccine via passive transfusion with CD4(+)/CD8(+) T lymphocytes.

Lanzhou Lamb derived Rotavirus (RV) Vaccine (namely LLR) for children is only used in China. Since there were no reports on evaluation of LLR, even the data of phase IV clinical trial, we proceed the evaluation of LLR through focusing on T-cell to investigate whether LLR could induce the potential function involving in protection as a vaccine. Four groups of nude mice were transfused with CD4(+)/CD8(+) T-cells isolated from LLR-immunized (primed) and LLR-unimmunized (naïve) mice via intraperitonea (i.p.) respectively. Consequently, the adoption mice were challenged with mice-origin wild rotavirus EDIM (Epizootic Diarrhea of Infant Mice) by intragastric administration. Series of fecal/serum samples were collected and viral shedding, then serum IgA/IgG and secreted IgA were assayed. Compared to the mice transfused with T lymphocytes from naïve mice, the nude mice transfused with CD4(+) T lymphocytes from primed mice induce fecal and serum IgA increasing more rapidly, and have a shorter duration of virus shedding too. Whereas, no significant difference in virus clearance was found between the mice transfused with CD8(+) T lymphocytes isolated from primed and naïve mice. Therefore, we cleared the distinct roles of transfused CD4(+)/CD8(+) T lymphocytes for rotavirus clearance in nude mice, that the viral clearance conducted by CD4(+) T lymphocytes. Meanwhile, it has ability to help induction of LLR specific immunogenicity. Comparing with the transfusion of cell from primed and naïve mice, LLR can induce CD4(+) T lymphocytes memory which is a potential index to reflect the immunogenicity and protection, while CD8(+) T lymphocytes remove rotavirus by CTL with little memory ability.

Hepatitis C Virus Subtype and Evolution Characteristic Among Drug Users, Men Who Have Sex With Men, and the General Population in Beijing, China.

The aim of this study was to characterize the current molecular epidemiology of hepatitis C virus (HCV) infection and evaluate the evolutionary patterns of HCV subtypes in Beijing, China, among different subpopulations.The whole blood samples and behavioral data were collected from a total of 10,354 subjects, including drug users (DUs), men who have sex with men (MSM), and the general population, in Beijing from 2010 to 2011. Samples were tested for HCV infection using both enzyme-linked immunosorbent assay (ELISA) and real-time PCR. All viremic subjects were then sequenced by nested PCR over core/E1 and NS5B regions. Phylogenetic and phylogeographic analysis was performed by BEAST software.In total, 217 subjects (2.1%) were tested positive for HCV by antibody or vRNA-based testing. HCV prevalence rates for DUs, MSM, and the general population were 26.2%, 0.54%, and 0.37%, respectively. The 156 HCV RNA-positive samples were sequenced. Nine HCV genotypes, including 1a, 1b, 2a, 3a, 3b, 6a, 6n, 6u and 6v, were detected. The most prevalent subtypes were 3b (36.09%), 1b (32.54%), and 3a (16.57%). Bayesian evolutionary analysis estimated that the time of introduction of subtype 1b into Beijing was 2004 (95% CI: 1997.7, 2007.7), with subtypes 3a and 3b being introduced later in 2006. Evolutionary analyses further suggested that subtype 1b from Beijing and Shanghai were closely related, whereas subtype 3a sequences were more similar with sequences from Yunnan, Guangzhou, Hong Kong, and Jiangsu. Subtype 3b sequences were closely related to those from Yunnan, Guangdong, and Hong Kong.Thus, the current HCV epidemic in Beijing is complex, heavily affecting DUs, and involving multiple genotypes that likely spread from different regions in China with its large migrant population.