Efficacy of recombinant Marek’s disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.     

Hepatitis B virus-like particles expressing Plasmodium falciparum epitopes induce complement-fixing antibodies against the circumsporozoite protein

The repetitive structure of compact virus-like particles (VLPs) provides high density displays of antigenic sequences, which trigger key parts of the immune system. The hepatitis B virus (HBV) and human papilloma virus (HPV) vaccines exploit the assembly competence of structural proteins, which are the effective immunogenic components of the prophylactic HBV and HPV vaccines, respectively. To optimize vaccine designs and to promote immune responses against protective epitopes, the “Asp-Ala-Asp-Pro” (NANP)-repeat from the Plasmodium falciparum circumsporozoite protein (CSP) was expressed within the exposed, main antigenic site of the small HBV envelope protein (HBsAgS); this differs from the RTS,S vaccine, in which CSP epitopes are fused to the N-terminus of HBsAgS. The chimeric HBsAgS proteins are assembly competent, produce VLPs, and provide a high antigenic density of the NANP repeat sequence. Chimeric VLPs with four or nine NANP-repeats (NANP4 and NANP9, respectively) were expressed in mammalian cells, the HBsAgS- and CSP-specific antigenicity of the VLPs was determined, and the immunogenicity of the VLPs assessed in relation to the induction of anti-HBsAgS and anti-CSP antibody responses. The chimeric VLPs induced high anti-CSP titres in BALB/c mice independent of the number of the NANP repeats. However, the number of NANP repeats influenced the activity of vaccine-induced antibodies measured by complement fixation to CSP, one of the proposed effector mechanisms for Plasmodium neutralization in vivo. Sera from mice immunized with VLPs containing nine NANP repeats performed better in the complement fixation assay than the group with four NANP repeats. The effect of the epitope-specific density on the antibody quality may instruct VLP platform designs to optimize immunological outcomes and vaccine efficacy.     

Development of a respiratory syncytial virus vaccine using human hepatitis B core-based virus-like particles to induce mucosal immunity

BackgroundRespiratory syncytial virus (RSV) is an important viral pathogen responsible for severe infection of the lower respiratory tract in children under the age of 5 years. No vaccines against RSV are currently in clinical use. Vaccine-associated enhanced respiratory disease (ERD) caused by excess Th2 type responses was observed in a clinical trial of formalin-inactivated RSV (FI-RSV) in antigen-naïve infants. Thus, inducing a balanced immune response is a crucial issue in the development of an RSV vaccine.MethodsIn this study, we constructed, expressed, and purified a recombinant RSV vaccine candidate (i.e., HRØ24) containing the two heptad repeat regions and the antigenic sites Ø, II, and IV of the RSV F protein. The RSV vaccine candidate was intranasally administrated to BALB/c and C57BL/6 mice in combination with virus-like particles (VLPs) derived from the core protein of the hepatitis B virus (HBc). Mucosal immunity to HRØ24 was then assessed.ResultsIntranasal administration of HBc VLPs in combination with HRØ24 induced serum IgGs against HRØ24 as well as lung HRØ24-specific sIgAs in both C57BL/6 and BALB/c mouse models. The secretion of IFN-γ from splenocyte re-stimulation and an elevated ratio of serum IgG2a to IgG1 indicated that the immune response induced by the HBc VLPs/HRØ24 mixture was Th1-biased. Weight loss of <5% and no to low eosinophil infiltration was observed in histological analysis of the lung following a challenge with the RSV A2 strain. These results suggest that the HBc VLPs/HRØ24 combination conferred substantial partial protection against RSV-induced illness in mice.ConclusionsLong-term immunity to RSV-induced illness was achieved via intranasal vaccination using a mixture of HBc VLPs and HRØ24 in mouse models.     

A Proposal for an Alternative Approach to Particle Size Method Development During Early-Stage Small Molecule Pharmaceutical Development

Particle size analysis in the pharmaceutical industry has long been a source of debate regarding how best to define measurement accuracy; the degree to which the result of a measurement or calculation conforms to the true value. Defining a “true” value for the size of a particle can be challenging as the output of its measurement will differ because of variations in measurement approaches, instrumental differences and calculation methods. Consequently, for “real” particles, a universal “true” value does not exist and accuracy is therefore not a definable characteristic. Accordingly, precision is then a measure of the ability to reproducibly achieve a measurement of unknown relevance.This article proposes, in place of accuracy, a means to define the “appropriateness” of a measurement in line with the critical quality attributes (CQA) of the material being characterized. The decision as to whether the measurement is correct should involve a link to the CQA; that is, correlation should be demonstrated, without which the measured particle size cannot be defined as a critical material attribute.Correspondingly, methods should also be able to provide sufficient precision to demonstrate discrimination relating to variation in the CQA. The benefits and challenges of this approach are discussed.     

Virus-like particle vaccine with B-cell epitope from porcine epidemic diarrhea virus (PEDV) incorporated into hepatitis B virus core capsid provides clinical alleviation against PEDV in neonatal piglets through lactogenic immunity

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope ⁷⁴⁸YSNIGVCK⁷⁵⁵ from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC).     

依据CLSI EP9-A3评价两种方法学检测糖类抗原242的可比性

目的评价两种方法学检测糖类抗原242(CA242)结果的可比性,以评估磁微粒化学发光法检测CA242是否能够满足临床的需求。方法根据美国临床和实验室标准协会(CLSI)新指南EP9-A3文件要求,收集2018年1-7月首都医科大学附属北京康复医院和北京大学首钢医院肿瘤患者检测剩余的新鲜血清标本100例,以Fujirebio Diagnostics AB的酶联免疫法为参比方法,安图生物的磁微粒化学发光法为评估方法,对2种方法检测CA242的结果进行方法学比对和偏移评估。选择Passing-Baklok回归方法进行线性拟合,采用Wilcoxon符号秩检验及Spearman相关分析。结果在4.31~295.63 U/ml范围内,2种方法学的CA242检测结果具有较好的相关性(r=0.991,截距0.652)。参比方法和评估方法比较,差异无统计学意义[(53.75±6.69)U/ml比(56.11±6.86)U/ml,t=0.246,P=0.806]。将CA242的医学决定水平25.00 U/ml代入选取的最佳回归模型拟合方程,计算得到的相对偏移3.52%,<1/2TEa±12.5%(TEa为国家卫生健康委临床检验中心室间质量评审允许总误差),满足要求。结论安图生物的磁微粒化学发光法和Fujirebio Diagnostics AB酶联免疫法检测CA242结果具有可比性,满足临床需要。     

Immune-pineal axis protects rat lungs exposed to polluted air

Environmental pollution in the form of particulate matter <2.5 μm (PM_2.5) is a major risk factor for diseases such as lung cancer, chronic respiratory infections, and major cardiovascular diseases. Our goal was to show that PM_2.5 eliciting a proinflammatory response activates the immune-pineal axis, reducing the pineal synthesis and increasing the extrapineal synthesis of melatonin. Herein, we report that the exposure of rats to polluted air for 6 hours reduced nocturnal plasma melatonin levels and increased lung melatonin levels. Melatonin synthesis in the lung reduced lipid peroxidation and increased PM_2.5 engulfment and cell viability by activating high-affinity melatonin receptors. Diesel exhaust particles (DEPs) promoted the synthesis of melatonin in a cultured cell line (RAW 264.7 cells) and rat alveolar macrophages via the expression of the gene encoding for AANAT through a mechanism dependent on activation of the NFκB pathway. Expression of the genes encoding AANAT, MT1, and MT2 was negatively correlated with cellular necroptosis, as disclosed by analysis of Gene Expression Omnibus (GEO) microarray data from the human alveolar macrophages of nonsmoking subjects. The enrichment score for antioxidant genes obtained from lung gene expression data (GTEx) was significantly correlated with the levels of AANAT and MT1 but not the MT2 melatonin receptor. Collectively, these data provide a systemic and mechanistic rationale for coordination of the pineal and extrapineal synthesis of melatonin by a standard damage-associated stimulus, which activates the immune-pineal axis and provides a new framework for understanding the effects of air pollution on lung diseases.     

Increases in HPV-16/18 antibody avidity and HPV-specific memory B-cell response in mid-adult aged men post-dose three of the quadrivalent HPV vaccine

Strong quantitative and functional antibody responses to the quadrivalent human papillomavirus (HPV) vaccine were reported in mid-adult aged men, but there are limited data on the avidity of the antibody response and the memory B-cell response following vaccination. Although circulating antibodies induced by vaccination are believed to be the main mediators of protection against infection, evaluation of avidity of antibodies and memory B cell responses are critical for a better understanding of the vaccine immunogenicity mechanisms. Both the modified enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunosorbent spot (ELISpot) assay are tools to measure the humoral and cellular immune responses post vaccination to characterize vaccine immunogenicity. The avidity of HPV-16 and HPV-18 specific IgG in the serum of mid-adult aged men (N = 126) who received three quadrivalent HPV vaccine doses was examined using a modified ELISA. HPV-16 memory B-cell responses were assessed via ELISpot at month 0 (prior to vaccination) and 1-month post-dose three of the vaccine (month 7). The quadrivalent vaccine induced an increase in HPV-16 and HPV-18 antibody avidity at month 7. HPV-18 avidity levels moderately correlated with anti-HPV-18 antibody titers, but no association was observed for HPV-16 antibody titers and avidity levels. The HPV-16-specific memory B-cell response was induced following three vaccine doses, however, no association with anti-HPV-16 antibody avidity was observed. Three doses of quadrivalent HPV vaccine increased antibody affinity maturation for HPV-16/18 and increased the frequency of anti-HPV-16 memory B-cells in mid-adult aged men.