Elevated homocysteine (Hcy) levels are well established as an independent risk factor for atherosclerosis and its associated cardiovascular diseases. Macrophage pyroptosis- mediated inflammation plays a crucial role in the progression of atherosclerosis. Notably, glycoprotein non-metastatic melanoma protein B (GPNMB) expression is increased in macrophages within atherosclerotic plaques; however, whether GPNMB participates in Hcy-induced macrophage pyroptosis remains elusive. In the present study, we found that GPNMB expression was upregulated in Hcy- treated THP-1- derived macrophages. Consistently, serum GPNMB levels were significantly higher in patients with hyperhomocysteinemia (HHcy) compared with healthy controls. Functional experiments showed that silencing GPNMB reduced Hcy-triggered pyroptosis in THP-1-derived macrophages, whereas GPNMB overexpression exerted the opposite effect. Mechanistically, GPNMB upregulated the NOX2/NF-κB signaling pathway in THP-1-derived macrophages. Importantly, the pro-pyroptotic effect of GPNMB overexpression in Hcy-treated THP-1-derived macrophages was counteracted by either inhibition of NADPH oxidase 2 (NOX2) using the specific inhibitor gp91ds-tat or blockade of NF-κB activation with the inhibitor BAY11-7082. Moreover, serum GPNMB levels were correlated with serum Hcy levels and lipid profiles in both healthy individuals and HHcy patients. Collectively, these findings demonstrate that GPNMB facilitates Hcy-induced macrophage pyroptosis associated with the upregulation of the NOX2/NF-κB signaling pathway, highlighting the potential relevance of GPNMB as a candidate target for the clinical management of HHcy-related atherosclerotic cardiovascular disease.

Acetaminophen (APAP)-induced liver injury (AILI) is a leading cause of acute liver failure, with limited preventive or therapeutic options. The role of betaine-homocysteine methyltransferase (BHMT), a key enzyme in the methionine cycle, remains unclear. We found that BHMT, primarily expressed in hepatocytes, showed reduced expression in the liver but elevated serum levels in the APAP-induced liver injury (AILI) mouse model. GalNAc-mediated targeted knockdown of Bhmt in hepatocytes aggravated AILI in mice. Through RNA-seq screening, we found that Bhmt deficiency dramatically suppressed stearoyl-coenzyme A desaturase 1 (SCD1) expression. Knockdown of Scd1 also exacerbated AILI. Mechanistically, Bhmt knockdown decreased the DNA methylation of BACH1 (BTB and CNC homology 1), a transcriptional factor, leading to upregulated BACH1 expression in primary mouse hepatocytes (PMHs) treated with APAP. BACH1 then bound to the enhancer region of Scd1, transcriptionally repressing SCD1. Lipidomic analysis revealed that Bhmt or Scd1 deficiency reduced levels of intracellular unsaturated fatty acids, particularly oleic acid (OA), whereas SCD1 overexpression increased OA levels and decreased lipid peroxides. OA administration alleviated AILI and mitigated the hepatotoxicity associated with Bhmt or Scd1 knockdown. Our findings indicate that BHMT mitigates AILI via the BACH1-SCD1-OA axis, suggesting that BHMT could serve as a preventive target for AILI, while increasing OA intake may offer dietary benefits for patients.

Ferroptosis is one of the most critical biological consequences of glutathione depletion. Excessive oxidative stress, indicated by an elevated oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio, is recognized as a key driver of ferroptosis. However, in glutathione depletion-induced ferroptosis, a marked decrease in total glutathione levels (including both GSH and GSSG) is frequently observed, yet its significance remains understudied. Protein S-glutathionylation (protein-SSG) levels are closely linked to the redox state and cellular glutathione pools including GSH and GSSG. To date, the role of protein-SSG during cell ferroptosis induced by glutathione depletion remains poorly understood. Here, we demonstrated that upregulation of CHAC1, a glutathione-degrading enzyme, acted as a key regulator of protein-SSG formation and exacerbated glutathione depletion-induced ferroptosis. This effect was observed in both in vitro and in vivo models, including erastin-induced ferroptosis across multiple cell lines and acetaminophen overdose-triggered ferroptosis in hepatocytes. Deficiency of CHAC1 resulted in increased glutathione pools, enhanced protein-SSG, improved liver function, and attenuation of hepatocyte ferroptosis upon acetaminophen challenge. These protective effects were reversed by CHAC1 overexpression. Using quantitative redox proteomics, we identified glutathione pool-sensitive S-glutathionylated proteins. As an important example, we discovered that ADP-ribosylation factor 6 (ARF6) was regulated by S-glutathionylation during glutathione depletion-induced ferroptosis. Our findings revealed that CHAC1 upregulation reduced the S-glutathionylation of ARF6, resulting in decreased ARF6 levels in lysosomes. This, in turn, enhanced the localization of the transferrin receptor (TFRC) on the cell membrane and increased transferrin uptake, ultimately compromising the protective role of ARF6 in ferroptosis induced by glutathione depletion. Targeting TFRC using GalNAc-siTfrc mitigated acetaminophen-induced liver injury in vivo. In conclusion, our study provide evidence that availability of glutathione pools affects protein S-glutathionylation and regulates protein functions to influence the process of ferroptosis, which opens an avenue to understanding the cell ferroptosis induced by glutathione depletion.

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.

目的 初步考察稳定表达胰蛋白酶原的重组MDCK细胞(即MTY6细胞)分离流感病毒的效果.方法 按照世界卫生组织(World Health Organization,WHO)全球流感监测网络和国家流感中心(National Influenza Centers,NICs)推荐的病毒分离方法,使用MDCK和MTY6细胞同步分离包含H1N1、H3N2和B型流感病毒核酸阳性的咽拭子样本共20份,对分离培养液分别使用豚鼠红细胞和鸡血红细胞进行凝集试验,统计比较不同类型红细胞凝集效果,以及不同细胞分离病毒阳性率和血凝素滴度.结果 相同病毒分离物对两种类型红细胞的凝集效果不同,豚鼠红细胞完全凝集时间约为鸡血红细胞2倍,沉积形状呈环状,血凝滴度平均为鸡红细胞的23.6±1.2倍.相同条件下,有3份样本两种细胞均分离阴性,11份样本两种细胞均分离阳性,其余6份样本MDCK细胞分离阴性,而MTY6细胞分离阳性,MTY6细胞分离阳性率较MDCK细胞高30％;分离阳性样本包括H1N1亚型8份,MTY6细胞分离病毒血凝素滴度显著高于MDCK细胞,平均为13.0(1.7,23.0)倍;H3N2亚型和B型各2份,MTY6细胞分离病毒血凝素滴度均高于MDCK细胞,平均为11.3和32.0倍.结论 综合考虑,对于细胞分离流感病毒检测,豚鼠红细胞优于鸡血红细胞.相同条件下,MTY6细胞较MDCK细胞分离流感病毒的灵敏度更高,具有作为流感病毒分离优质细胞基质的潜力.

This article reviews the relevant studies on the efficacy and safety of influenza, pneumococcal and COVID-19 vaccination among tumor patients worldwide in recent years. By combing and analyzing the retrieved literature, the results show that influenza and pneumococcal vaccination can significantly reduce the morbidity and hospitalization rate of infectious diseases in tumor patients, reduce the risk of cardiovascular events and death, and significantly improve survival prognosis. COVID-19 vaccination can also protect tumor patients, especially those who have completed full dose vaccination. Authoritative guidelines and consensuses worldwide all recommend that tumor patients receive influenza, pneumococcal and COVID-19 vaccines. We should carry out relevant researches, as well as take effective measures to strengthen patient education, so that tumor patients can fully experience the health protection brought by the vaccine to this specific group.

Objective: To evaluate the immunogenicity and safety of revaccination of 23-valent pneumococcal polysaccharide vaccine (PPV23) in elderly people aged ≥60 years. Methods: The elderly aged ≥60 years with 1 dose of PPV23 vaccination were selected as revaccination group and those without history of pneumococcal vaccine immunization were selected as the first vaccination group. One dose of PPV23 was administered to both groups, and the first blood samples were collected before vaccination while the second blood samples were collected on day 28-40 after vaccination. ELISA was used to detect the concentrations of anti-specific serotype Streptococcus pneumoniae podocyte polysaccharide immunoglobulin G, and the safety of the vaccination was evaluated after 30 days. Results: The geometric mean concentration (GMC) of antibody to 23 serotypes before the vaccination (0.73-13.73 μg/ml) was higher in revaccination group than in the first vaccination group (0.39-7.53 μg/ml), the GMC after the vaccination (1.42-31.65 μg/ml) was higher than that before the vaccination (0.73-13.73 μg/ml) in the revaccination group, and the GMC after the vaccination (1.62-43.76 μg/ml) was higher than that before the vaccination (0.39-7.53 μg/ml) in the first vaccination group; the geometric mean growth multiple in revaccination group (2.16-3.60) was lower than that in the first vaccination group (3.86-16.13); The mean 2-fold antibody growth rate was lower in revaccination group (53.68%, 95%CI: 52.30%-55.06%) than in the first vaccination group (93.16%, 95%CI: 92.18%- 94.15%), all differences were significant (P<0.001). After the vaccination, 13 serotypes of GMC were higher in the first vaccination group than in revaccination group (P<0.001), the differences were not significant for 10 serotypes of GMC (P>0.05). The incidence of local adverse reaction was 19.20% and 13.27% in revaccination group and the first vaccination group, respectively (P=0.174). Conclusions: The antibody level in ≥60 years people who received one dose of PPV23 after a 5-year interval was still higher than that in unvaccinated people. The antibody level decreased after 5 years of the first vaccination, and the antibody level could be rapidly increased by one more dose vaccination, but the overall immune response was lower than that of the first vaccination; revaccination with PPV23 has a good safety.

目的 对H5N1型流感病毒灭活疫苗(MDCK细胞)原液蛋白成分进行分析,为完善该疫苗的质量控制方法及疫苗的研发提供依据.方法 将H5N1型流感病毒株接种于MDCK细胞,病毒培养48 h后,收获病毒液,经澄清、超滤浓缩、β-丙内酯灭活、Capto Q和Sephorase 4FF两步层析纯化,获得原液样品.应用透射电镜分析样品中病毒颗粒的形态;单向免疫扩散(single radial immunodiffusion,SRID)试验鉴别病毒原液血凝素(hemagglutinin,HA);高效液相色谱进行纯度分析;梯度SDS-PAGE进行蛋白电泳分析;液质联用仪(LC/MS)结合对回收的各条带蛋白多肽进行二级质谱序列测定的方法分析样品中所含的蛋白成分.结果 在透射电镜下可观察到80～120 nm球形H5N1流感病毒颗粒,呈典型的流感病毒形态;鉴别试验表明病毒原液抗原性与病毒株相一致;经梯度SDS-PAGE分析,病毒原液有2条带;LC/MS质谱分析显示,原液样品中H5N1型流感病毒蛋白为主要成分,包括H5N1型流感病毒的NP、M1、HA、NA等蛋白.结论 对H5N1型流感病毒灭活疫苗制备过程中原液的蛋白成分进行了分析,为该类疫苗的研发及质量控制提供了参考依据.

With the continuous in-depth study of the interaction mechanism between viruses and hosts, the virus has become a promising tool in cancer treatment. In fact, many oncolytic viruses with selectivity and effectiveness have been used in cancer therapy. Human enterovirus is one of the most convenient sources to generate oncolytic viruses, however, the high seroprevalence of some enteroviruses limits its application which urges to exploit more oncolytic enteroviruses. In this study, coxsackievirus B5/Faulkner (CV-B5/F) was screened for its potential oncolytic effect against non-small cell lung cancers (NSCLCs) through inducing apoptosis and autophagy. For refractory NSCLCs, DNA-dependent protein kinase (DNA-PK) or ataxia telangiectasia mutated protein (ATM) inhibitors can synergize with CV-B5/F to promote refractory cell death. Here, we showed that viral infection triggered endoplasmic reticulum (ER) stress-related pro-apoptosis and autophagy signals, whereas repair for double-stranded DNA breaks (DSBs) contributed to cell survival which can be antagonized by inhibitor-induced cell death, manifesting exacerbated DSBs, apoptosis, and autophagy. Mechanistically, PERK pathway was activated by the combination of CV-B5/F and inhibitor, and the irreversible ER stress-induced exacerbated cell death. Furthermore, the degradation of activated STING by ERphagy promoted viral replication. Meanwhile, no treatment-related deaths due to CV-B5/F and/or inhibitors occurred. Conclusively, our study identifies an oncolytic CV-B5/F and the synergistic effects of inhibitors of DNA-PK or ATM, which is a potential therapy for NSCLCs.

Objective:To explore the effect of actual multiplicity of infection(MOI) on virus proliferation in large scale production of rotavirus vaccine.Methods:The titer of rotavirus inoculation solution was detected with fluorescent focus assay when inoculated in Vero cells. Actual MOI was calculated as quantity of virus divided by quantity of cells. Cytopathic effect time and single harvest titer were monitored to reflect the effect of actual MOI on rotavirus proliferation in large scale production.Results:In large scale production, MOI in the range of 0.002-0.074 affected the course of cytopathic effect, and showed a weak correlation with the proliferation of rotavirus. The titers of single rotavirus harvest were all above 7.0 lg fluorescent focus unit(FFU)/ml, meeting the quality standard (≥6.5 lgFFU/ml).Conclusion:Actual MOI shows a weak correlation with the proliferation of rotavirus in large scale production.

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

论文对流感疫苗、肺炎球菌疫苗等非国家免疫规划疫苗的国内外接种现状及有效提高疫苗接种率的相关研究进行综述.通过对检索文献进行梳理和分析,结果显示,部分国家流感、肺炎球菌等疫苗接种率处于较高水平,原因在于国家将其纳入免疫规划或给予政府财政支持,此外,一系列整合患者教育、医护培训、电子信息技术、强制接种等的多模式策略也有效提高了疫苗接种率.目前国内流感和肺炎球菌等疫苗接种率整体偏低,应积极借鉴各国的先进策略和经验,采取有效措施,以提高疫苗接种率,更好的保障人群生命与健康.

目的 建立用于检测流感疫苗神经氨酸酶(neuraminidase,NA)活性的荧光底物法,并进行验证.方法 以唾液酸类似物4-MUNANA作为酶底物,通过测定NA催化底物分解后产物的荧光强度来评价NA活性.选取基于MDCK细胞制备的四价流感疫苗各单价原液(H1N1、H3N2、BV和BY)作为样品,优化荧光底物法的底物酶浓度(10、20、30、40、50 μmol/L)、作用时间(每 2 min 检测 1 次,至 90 min)、缓冲液 pH(4.5、5.5、6.5、7.5、8.5 和 9.5)及反应温度(4、25、37和45℃).以已知活性的重组NA蛋白作为参考品,验证方法的线性范围、准确度及精密度.用优化的方法检测流感疫苗生产过程中的样品(基于MDCK细胞制备的H1N1、H3N2、BV、BY相应的病毒收获液、纯化液和原液)及以MDCK细胞基质和鸡胚基质制备的四价流感裂解疫苗成品各1批.结果 最佳反应条件为:酶底物浓度20μmol/L,反应时间40 min,pH 7.5,反应温度37℃.重组NA蛋白参考品浓度在62.5～2 000 U/L范围内与荧光强度呈良好的线性关系,R2＞0.99;1 600、800、400 U/L 3个浓度参考品的回收率分别为92.60％～106.96％、85.54％～113.11％、87.00％～114.54％,于 2 个时间点共 12 次检测结果的 CV 分别为 6.40％、8.25％、6.92％.H1N1、H3N2、BV 和 BY 4种亚型的流感疫苗半成品在不同阶段NA活性逐渐降低;细胞基质及鸡胚基质四价流感裂解疫苗成品的NA活性分别为61 585.82和69 583.51 U/L.结论 成功建立并优化了一种NA活性的荧光底物检测方法,该方法具有良好的准确度和精密度,可应用于流感疫苗生产过程及成品中NA活性的检测.

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

目的 建立用于定量检测流感疫苗神经氨酸酶(neuraminidase,NA)含量的双抗体夹心ELISA方法,并进行验证.方法 采用多肽合成方式合成NA保守序列,经皮下多点免疫日本大耳白兔和豚鼠,共免疫5次,末次免疫后2周分别经颈动脉和心脏采血,分离血清,通过Protein G/A层析纯化,制备NA通用抗体.以鼠源通用抗体作为包被抗体,兔源通用抗体经HRP标记后作为酶标抗体,建立双抗体夹心ELISA法.确定包被抗体(32、16、8、4、2、1 μg/mL)的工作浓度及酶标抗体(1 ∶ 50～1 ∶ 6 400)的稀释度.验证方法的线性范围、准确度、重复性、中间精密度及耐用性.采用建立的方法测定流感裂解疫苗H1N1、H3N2、BV和BY型单价原液中的NA含量,并与荧光底物法进行比较.结果 兔源和鼠源通用抗体的效价分别为128 000和64 000,纯度分别为95％和96％,蛋白浓度分别为2 339和1 780μg/mL.建立双抗体夹心ELISA法的最佳包被抗体工作浓度为16μg/mL,最佳酶标抗体稀释度为1:400.NA(H3N2型)参考品在20～640 ng/mL浓度范围内与A450呈良好线性关系,R2均＞0.99;500、200、50 ng/mL的NA(H3N2型)参考品的平均样品回收率分别为102.15％、100.89％、100.70％,重复6次检测结果的CV均＜10％,2名实验员3次检测结果的CV均＜15％;不同抗原反应时间及酶标二抗孵育时间的样品回收率为85.41％～103.81％.建立的双抗体夹心ELISA法检测流感裂解疫苗H1N1、H3N2、BV、BY型单价原液NA含量分别为8.06、13.20、6.93、6.18 μg/mL,荧光底物法检测的NA活性分别为29 833、36 800、28 907、25 871 U/L,两者结果呈正相关(R2=0.979 2).结论 建立的NA含量双抗体夹心ELISA定量检测法具有良好的准确度、重复性、中间精密度和耐用性,可用于流感疫苗的检定及生产过程中对NA含量的质量控制.

目的 制备甲型H1N1流感病毒mRNA疫苗,并评价其免疫原性.方法 设计并构建分别含有甲型H1N1流感病毒血凝素(hemagglutinin,HA)和绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的模板质粒,通过单酶切反应制备线性化转录模板,体外转录合成HA-mRNA和EGFP-mRNA.EGFP-mRNA体外转染HEK293T细胞,荧光显微镜下观察荧光表达情况.利用纳米药物制备系统制备HA-mRNA脂质体纳米颗粒(lipid nanoparticles,LNPs),即HA-mRNA-LNPs,测量粒径并计算包封率.用含2和10 μg HA-mRNA的HA-mRNA-LNPs经肌肉分别免疫BALB/c小鼠,同时设LNPs组(等体积未包裹mRNA的LNPs)和阴性对照组(等体积PBS),3周后进行加强免疫,免疫剂量和途径同上.加强免疫后3周,经小鼠眼眶后静脉丛采血,分离血清,采用血凝抑制(haemagglutination inhibition,HI)试验和病毒微量中和(microneutralization assay,MN)试验分别检测血凝抑制抗体滴度及中和抗体水平,并计算几何平均滴度(geometric average titer,GMT).结果 EGFP-mRNA转染HEK293T细胞后,镜下可见明显EGFP表达.HA-mRNA-LNPs的颗粒平均直径为70.2 nm,聚合物分散性指数为0.15,包封率约为80％.与阴性对照组比较,LNPs组小鼠血清的HI GMT和MN GMT差异均无统计学意义(P＞0.05),2和10 μg剂量组均显著升高(P均＜0.000 1);10 μg剂量组均显著高于2 μg剂量组(P均＜0.01).结论 制备的甲型H1N1流感病毒mRNA疫苗在BALB/c小鼠模型中具有较好的免疫原性,本研究为流感mRNA候选疫苗的研发提供了实验依据.

目的 探讨在小鼠模型中H5N1型流感病毒神经氨酸酶(neuraminidase,NA)的免疫原性,并进行攻毒试验评价其免疫保护作用.方法 制备H5N1型流感病毒裂解疫苗原液,磁珠法纯化获取NA蛋白.将BALB/c小鼠分为 2 μg NA、10 μg NA、50 μg NA、2 μg NA+MF59、10 μg NA+MF59、50 μg NA+MF59 和 MF59 组,共 7 组,每组10只,肌内注射免疫.初次免疫2周后,相同程序/剂量进行加强免疫,第4周采血分离血清,ELISA法测定效价;第5周进行攻毒试验,监测攻毒后2周内小鼠体重和体温变化,同时观察临床症状.结果 磁珠法纯化的H5N1 NA纯度达90％,浓度为201.23 μg/mL.2次免疫后,各剂量组血清效价呈剂量依赖性,加MF59佐剂组血清抗体效价均高于未加MF59佐剂组,其中50 μg NA+MF59组效价最高(平均效价为121 600).攻毒试验发现,50 μg NA+MF59能够保护100％(8只)的小鼠免受H5N1攻击,攻毒后50 μg NA+MF59组临床评分低于MF59组,但两组小鼠体温变化差异无统计学意义(P＞0.05).结论 免疫H5N1 NA能够诱导BLAB/c小鼠产生NA特异性抗体,使用MF59佐剂能够显著提高抗体效价,保护小鼠免受半数致死剂量的H5N1攻击,并能明显减轻临床症状.

目的 探索未成熟的小鼠骨髓来源树突状细胞(bone marrow derived dendritic cells,BMDCs)的分离诱导培养方法,并研究水包油佐剂对BMDCs蛋白表达的影响.方法 解剖小鼠获得小鼠骨髓细胞,分析在骨髓细胞诱导培养获得未成熟的BMDCs的过程中,红细胞裂解液(red blood cell lysis buffer,RBC)、诱导剂IL-4、rmGM-CSF及培养容器(6孔板或细菌培养皿)对获得未成熟BMDCs的纯度和成熟度的影响.用水包油佐剂AddaVax刺激培养收获的BMDCs 24 h后,检测BMDCs表面分子的表达差异,以及通过蛋白组学方法检测BMDCs细胞所有蛋白的表达差异.结果 一只小鼠的骨髓细胞诱导培养6 d,培养条件为无RBC处理、细菌培养皿培养,获得的BMDCs细胞数最高,为1.2×107/只,且BMDCs细胞的纯度较高(约60％),与在6孔板中IL-2和rmGM-CSF共同诱导获得的BMDCs纯度差异无统计学意义(P＞0.05).各种培养方法诱导BMDCs表面成熟标志CD86、CD40的表达分别＜40％、＜70％.水包油佐剂AddaVax诱导BMDCs表面分子MHC Ⅱ、CD40、CD80和CD86的表达均＞80％.蛋白组学分析显示,AddaVax 促进了 BMDCs 生物调节以及免疫应答相关蛋白 NRF2、KEAP1、P16、P53、Rb、BGFR、Ras、Raf、TRADD、ARIDIA、BAF155、ARID2、SWI/SNF的表达.结论 分离的骨髓细胞不需要进行RBC处理,在细菌培养皿用rmGM-CSF 诱导培养骨髓细胞6 d即可制备大量未成熟的BMDCs.水包油佐剂AddaVax促进了未成熟BMDCs的增殖、分化、成熟,并且促进了 BMDCs免疫相关通路蛋白的表达.

北京生物制品研究所有限责任公司研发生产的新型冠状病毒灭活疫苗(Vero细胞,BIBP-CorV疫苗)相继完成了 Ⅰ/Ⅱ、Ⅲ期临床试验,广泛应用于多个国家和地区.本文对BIBP-CorV疫苗在各国开展的真实世界研究进展作一综述,回顾总结了目前已报道的BIBP-CorV疫苗的真实世界研究数据,包括一般人群、高风险人群和特殊人群接种疫苗的有效性(vaccine effectiveness,VE)、免疫原性和安全性,为BIBP-CorV疫苗在大样本量的广泛人群中使用提供参考.