The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants

Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNγ and low frequencies of CD4(+) T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B- and T cells.     

Impact of influenza vaccine formulation with a detailed analysis of the cytokine response

Vaccination provides the most effective method of limiting the impact of influenza. Inactivated influenza vaccines are available in three formulations and more information needs to be generated on how antigen presented in different vaccine formulations influences the subsequent immune response. In the present study, we have investigated the effect of two different influenza vaccine formulations on the resulting antibody and cytokine responses and compared these responses with influenza infection. Mice were vaccinated intramuscularly with one or two doses of whole or split virus vaccine or alternatively intranasally infected with influenza virus. Lymphocytes were isolated from spleen cells and stimulated in vitro for 24 or 72 h for analysis of cytokine profile at the gene expression and at the protein level. Additionally, whole blood was collected and the serum antibody response investigated by haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA). We found that one dose of whole virus vaccine induced higher antibody and cytokine responses and thus was more immunogenic in unprimed mice than split virus vaccine. Whole virus vaccine induced a strong IFN-gamma (type 1) immune response after one dose of vaccine and a more mixed cytokine response after the second dose. Split virus vaccine induced a type 2 response, particularly after two vaccine doses. Our results show that two doses of vaccine (both vaccine formulation) were more effective in induction of Th2 type of cytokines and thus indicate that both the formulation and also the number of vaccine doses substantially influences the magnitude and quality of the immune response.     

A cell‐based H7N1 split influenza virion vaccine confers protection in mouse and ferret challenge models

Background In recent years, several avian influenza subtypes (H5, H7 and H9) have transmitted directly from birds to man, posing a pandemic threat. Objectives We have investigated the immunogenicity and protective efficacy of a cell based candidate pandemic influenza H7 vaccine in pre‐clinical animal models. Methods Mice and ferrets were immunised with two doses of the split virus vaccine (12–24 μg haemagglutinin) with or without aluminium hydroxide adjuvant and challenged 3 weeks after second dose with the highly pathogenic A/chicken/Italy/13474/99 (H7N1) virus. The H7N1‐specific serum antibody response was also measured. After challenge, viral shedding, weight loss, disease signs and death (only mice) were recorded. Results Low‐to‐modest serum antibody titres were detected after vaccination. Nevertheless, the vaccine induced significant protection from disease after challenge with the wild‐type virus. In the murine lethal challenge model, vaccination effectively prevented death and, furthermore, formulation with adjuvant reduced excessive weight loss and viral shedding. In ferrets, vaccination reduced viral shedding and protected against systemic spread of the virus. Conclusions We have extended to the H7 subtype the finding that protective efficacy may not be directly correlated with the pre‐challenge levels of serum antibodies, a finding which could be of great importance in assessing the potential effectiveness of pandemic influenza vaccines.     

异种骨支架材料的制备及其理化性能

目的:对比观察猪骨支架材料与同种异体骨支架材料的理化性能及力学性能。方法:实验于2006-03/12在南方医科大学人体解剖教研室(暨南方医科大学临床解剖学研究所和广东省组织构建与检测重点试验室)完成。实验材料:低温深冻6个月的6只成年猪(雌雄各3只)和4具新鲜健康成人尸体(男女各2具,由广州市红十字会南方医科大学遗体捐献接收点提供,家属知情同意)髂骨。实验方法:剔除软组织,刮除骨髓和骨膜,用锯骨机将松质骨切成5mm×5mm×40mm左右的骨条,超声清洗、H2O2和乙醇浸泡、甩干、冻干、辐照处理得到异种骨支架材料和同种异体骨支架材料。实验评估:①对2种材料进行扫描电镜观察。②对比2种材料孔隙率、蛋白质和钙磷含量及力学性能。结果:①扫描电镜观察结果:2种材料均具有骨本身的骨小梁、小梁间隙及骨内管腔系统,具有天然网状结构。三维支架系统形态完整。其中猪源性异种骨支架材料较人同种异体骨支架材料具有更多的三维孔隙,2种材料的孔隙大小接近,均在400μm左右。②材料蛋白质、钙磷含量及力学性能检测结果:异种骨支架材料的孔隙率高于同种异体骨[(57.20±1.37)%,(53.21±1.63)%,P<0.005],但蛋白含量低于同种异体骨[(23.36±0.48)%,(26.50±0.23)%,P<0.005],钙、磷含量与同种异体骨比较差异无显著性[钙:(1.7×105),(1.8×105)μg/g;磷:(1.0×105),(1.0×105)μg/g],2种材料弹性模量差异无显著性。结论:猪源性异种骨支架材料在理化性能方面与人骨支架材料极相近,可为骨细胞的生成提供基本的保证。     

The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine

Please cite this paper as: Svindland et al. The mucosal and systemic immune responses elicited by a chitosan‐adjuvanted intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses DOI:10.1111/j.1750‐2659.2011.00271.x. Background Development of influenza vaccines that induce mucosal immunity has been highlighted by the World Health Organisation as a priority (Vaccine 2005;23:1529). Dose‐sparing strategies and an efficient mass‐vaccination regime will be paramount to reduce the morbidity and mortality of a future H5N1 pandemic. Objectives This study has investigated the immune response and the dose‐sparing potential of a chitosan‐adjuvanted intranasal H5N1 (RG‐14) subunit (SU) vaccine in a mouse model. Methods Groups of mice were intranasally immunised once or twice with a chitosan (5 mg/ml)‐adjuvanted SU vaccine [7·5, 15 or 30 μg haemagglutinin (HA)] or with a non‐adjuvanted SU vaccine (30 μg HA). For comparison, another group of mice were intranasally immunised with a whole H5N1 (RG‐14) virus (WV) vaccine (15 μg HA), and the control group consisted of unimmunised mice. Results The chitosan‐adjuvanted SU vaccine induced an immune response superior to that of the non‐adjuvanted SU vaccine. Compared with the non‐adjuvanted SU group, the chitosan‐adjuvanted SU vaccine elicited higher numbers of influenza‐specific antibody‐secreting cells (ASCs), higher concentrations of local and systemic antibodies and correspondingly an improved haemagglutination inhibition (HI) and single radial haemolysis (SRH) response against both the homologous vaccine strain and drifted H5 strains. We measured a mixed T‐helper 1/T‐helper 2 cytokine response in the chitosan‐adjuvanted SU groups, and these groups had an increased percentage of virus‐specific CD4⁺ T cells producing two Thelper 1 (Th1) cytokines simultaneously compared with the non‐adjuvanted SU group. Overall, the WV vaccine induced higher antibody concentrations in sera and an HI and SRH response similar to that of the chitosan‐adjuvanted SU vaccine. Furthermore, the WV vaccine formulation showed a stronger bias towards a T‐helper 1 profile than the SU vaccine and elicited the highest frequencies of CD4⁺ Th1 cells simultaneously secreting three different cytokines (INFγ⁺, IL2⁺ and INFα⁺). As expected, two immunisations gave a better immune response than one in all groups. The control group had very low or not detectable results in the performed immunoassays. Conclusion The cross‐clade serum reactivity, improved B‐ and T‐cell responses and dose‐sparing potential of chitosan show that a chitosan‐adjuvanted intranasal influenza vaccine is a promising candidate vaccine for further preclinical development.     

肺纤维化模型大鼠肺组织中转化生长因子β1及转化生长因子β1mRNA的表达及黄芪莪术合剂的干预效应

目的:分析中药黄芪莪术合剂对博莱霉素所致大鼠肺间质纤维化的干预作用。方法:实验于2002-10/2003-02在天津中医药大学动物实验室完成。①健康SD大白鼠60只,正常饲养1周后,随机分为正常对照组6只、模型组18只、黄芪莪术合剂组18只、强的松组18只。②经气管滴入博莱霉素复制肺纤维化大鼠模型,黄芪莪术合剂组给予40.5mg/L黄芪莪术合剂,强的松组给予0.63g/L强的松盐水溶液,均采用灌胃方式给药,每次1.5mL,1次/d。正常对照组、模型组给予等量生理盐水。③模型组、黄芪莪术合剂组、强的松组分别于造模第3,7,21天每组6只麻醉下取右肺下叶组织后处死,正常对照组于第3天麻醉下取右肺下叶组织后处死。④采用免疫组织化学方法和原位杂交方法检测肺组织转化生长因子β1及转化生长因子β1mRNA的表达。结果:进入结果分析大鼠55只,中途脱落5只。①转化生长因子β1表达:造模第3,7,21天模型组高于正常对照组(P<0.01),黄芪莪术合剂组和强的松组明显低于模型组(P<0.01～0.05)。造模第3,21天强的松治疗组低于黄芪莪术合剂治疗组(P<0.05)。②转化生长因子β1mRNA表达:造模第3,7,21天模型组高于正常对照组,黄芪莪术合剂组和强的松组明显低于模型组(P<0.01);第7天时,强的松治疗组低于黄芪莪术合剂治疗组,第21天时黄芪莪术合剂治疗组低于强的松治疗组,差异均有显著性意义(P<0.05)。结论:中药黄芪莪术合剂具有显著抑制博莱霉素致大鼠肺纤维化的作用,抑制转化生长因子β1及转化生长因子β1mRNA的表达是其可能的机制之一。     

恒磁场对大鼠主动脉平滑肌细胞基质金属蛋白酶的影响

目的:观察不同磁感应强度恒磁场对培养的大鼠主动脉血管平滑肌细胞基质金属蛋白酶2活性的影响,以探讨磁场是否能用于经皮冠状动脉支架介入治疗术后再狭窄的防治。方法:实验于2005-12/2006-08在解放军第四军医大学西京医院心内科实验室完成。取纯种雄性SD大鼠,体质量200～250g,用含体积分数为0.1小牛血清的DMEM培养液体外培养大鼠主动脉血管平滑肌细胞,实验随机分为对照组,1Gs恒磁场组、5Gs恒磁场组、10Gs恒磁场组、50Gs恒磁场组,其中对照组不予磁场干预,其他各组分别给予磁场干预继续培养48h。运用基质金属蛋白酶2活性酶图分析法结合吸光度扫描分析,观察恒磁场对血管平滑肌细胞的基质金属蛋白酶2表达的影响。结果:与对照组相比,各组基质金属蛋白酶2的活性均明显被抑制,差异有显著性意义[(831.25±2.38)×102,(690.57±2.57)×102,(574.35±1.98)×102,(401.05±1.96)×102,(316.23±3.24)×102,P<0.05],不同磁场强度组组间分析显示具有剂量依赖性,随磁场强度加大,抑制作用也增强。结论:适当强度的恒磁场抑制血管平滑肌细胞的基质金属蛋白酶2活性的表达,磁场对经皮冠状动脉支架介入治疗术后的再狭窄可能具有防治作用。     

Differential north to south gastric cancer-duodenal ulcer gradient in China

There are suggestions that duodenal ulcer protects individuals from gastric cancer and that rice is ulcerogenic while wheat is gastro-protective. We aimed to examine the relationship of gastric cancer, duodenal and gastric ulcers in different geographical regions in China and identified dietary risk factors for duodenal ulcer and gastric cancer. The prevalence of peptic ulcer and gastric cancer among symptomatic patients in eight major cities, four each from the north and the south representing all the six defined regions of China were studied. Endoscopy and case records over a 10 year period were reviewed and cases of confirmed duodenal and gastric ulcer and gastric cancer, together with the total number of endoscopies performed per year, were recorded. Rates were expressed as cases/1000 endoscopies. Results were compared to another epidemiological study on diet and mortality in the same regions in China conducted at the same time. Duodenal ulcer rates were 2.4-fold higher in southern China than northern China, whereas gastric cancer rates were 1.6-fold higher in the north than in the south. Correlation studies showed for the first time an inverse linear relationship between the gastric cancer rates and the duodenal ulcer rates (r=-0.8076, P=0.015), as well as the duodenal ulcer: gastric ulcer ratios (r=-0.9133, P=0.002). Gastric ulcer rates were higher in southern China but did not correlate with the gastric cancer rates (r=0.1455, P=0.731). Duodenal ulcer rates were found to be related to daily rice intake (r=0.8554, P=0.029) and inversely related to daily wheat flour intake (r=-0.8472, P=0.033). Gastric cancer rates were not related to any dietary risk factors tested. We concluded there was an inverse relationship between gastric cancer rates and duodenal ulcer rates. Although duodenal ulceration and gastric cancer are both linked to Helicobacter pylori infection, the findings of this study indicate independent additional aetiological factors for the pathogenesis of these conditions. Dietary factors such as rice or wheat intake may play a role.     

Hypercholesterolemia promotes early renal dysfunction in apolipoprotein E-deficient mice

Background

Aging and dyslipidemia are processes which can lead to deleterious consequences to renal function. Therefore, the aim of this study was to determine the effects of both hypercholesterolemia and aging on renal function in mice.

Methods

Male hypercholesterolemic apolipoprotein E-deficient mice (ApoE, n = 13) and age-matched C57BL/6 control mice (C57, n = 15) were studied at 2 (young) and 8 (adult) month-old. At each time point, animals were placed in metabolic cages for 24 hours to urine volume and urinary creatinine quantification. Blood samples were collected for serum cholesterol, urea and creatinine measurements. Glomerular filtration rate (GFR) was estimated through creatinine clearance determination. Mesangial expansion was evaluated by Periodic Acid Schiff staining, renal fibrosis was determined through Masson's trichrome staining and neuronal nitric oxide synthase (nNOS) expression in the kidney was performed by Western Blotting. To statistical analysis two-way ANOVA followed by Fisher's post hoc test was used.

Results

Total plasma cholesterol was increased about 5-fold in ApoE mice at both time points compared to C57 animals. At 2-month-old, GFR was already markedly reduced in ApoE compared to C57 mice (187 ± 28 vs 358 ± 92 μL/min, p < 0.05). Adult C57 (-77%) and ApoE (-50%) mice also presented a significant reduction of GFR. In addition, serum urea was significantly increased in young ApoE animals compared to C57 mice (11 ± 1.3 vs 7 ± 0.9 mmol/L, p < 0.01). A significant mesangial expansion was observed at 2-month old ApoE mice compared to C57 mice (35 ± 0.6 vs 30 ± 0.9%, respectively, p < 0.05), which was aggravated at 8-month old animals (40 ± 3 and 35 ± 3%, respectively). Tubulointersticial fibrosis was augmented at both young (17 ± 2%, p < 0.05) and adult (20 ± 1%, p < 0.05) ApoE mice compared to respective C57 age controls (8 ± 1 and 12 ± 2%, respectively). The expression of nNOS was markedly reduced in a time-dependent manner in both strains.

Conclusions

These data show that both hypercholesterolemia and aging contribute to the loss of renal function in mice.