Folding domains within the ricin toxin A subunit as targets of protective antibodies

Efforts to develop an effective vaccine against ricin are focused on the engineering of attenuated and stable recombinant forms of the toxin's enzymatic A subunit (RTA). While several candidate antigens are in development, vaccine design and efficacy studies are being undertaken in the absence of a fundamental understanding of those regions of RTA that are critical in eliciting protective immunity. In this present study, we produced and characterized a collection of monoclonal antibodies (MAbs) directed against five distinct immunodominant regions on RTA, and used these MAbs to identify several key neutralizing epitopes on the toxin. Protective MAbs were directed against α-helices located in RTA folding domains 1 and 2, whereas non-neutralizing antibodies recognized random coils and loops that were primarily confined to folding domain 3. These data offer insights into the immunodominant and structural determinants on RTA that give rise to protective immunity, and for the first time provide an immunological rationale for ricin vaccine design.     

Thermal stability and epitope integrity of a lyophilized ricin toxin subunit vaccine

Biodefense vaccine are destined to be stockpiled for periods of time and deployed in the event of a public health emergency. In this report, we compared the potency of liquid and lyophilized (thermostabilized) formulations of a candidate ricin toxin subunit vaccine, RiVax, adsorbed to aluminum salts adjuvant, over a 12-month period. The liquid and lyophilized formulations were stored at stressed (40 °C) and unstressed (4 °C) conditions and evaluated at 3, 6 and 12-month time points for potency in a mouse model of lethal dose ricin challenge. At the same time points, the vaccine formulations were interrogated in vitro by competition ELISA for conformational integrity using a panel of three monoclonal antibodies (mAbs), PB10, WECB2, and SyH7, directed against known immunodominant toxin-neutralizing epitopes on RiVax. We found that the liquid vaccine under stress conditions declined precipitously within the first three months, as evidenced by a reduction in in vivo potency and concomitant loss of mAb recognition in vitro. In contrast, the lyophilized RiVax vaccine retained in vivo potency and conformational integrity for up to one year at 4 °C and 40 °C. We discuss the utility of monitoring the integrity of one or more toxin-neutralizing epitopes on RiVax as a possible supplement to animal studies to assess vaccine potency.     

Vaccine-induced intestinal immunity to ricin toxin in the absence of secretory IgA

The RNA N-glycosidase ribosome inactivating proteins (RIPs) constitute a ubiquitous family of plant- and bacterium-derived toxins that includes the category B select agents ricin, abrin and shiga toxin. While these toxins are potent inducers of intestinal epithelial cell death and inflammation, very little is known about the mechanisms underlying mucosal immunity to these toxins. In the present study, we report that secretory IgA (SIgA) antibodies are not required for intestinal immunity to ricin, as evidenced by the fact that mice devoid of SIgA, due to a mutation in the polymeric immunoglobulin receptor, were impervious to the effects of intragastric toxin challenge following ricin toxoid immunization. Furthermore, parenteral administration of ricin-specific monoclonal IgGs, directed against either ricin's enzymatic subunit (RTA) or ricin's binding subunit (RTB), to wild type mice was as effective as monoclonal IgAs with comparable specificities in imparting intestinal immunity to ricin. These data are consistent with reports from others demonstrating that immunization of mice by routes known not to induce mucosal antibody responses (e.g., intramuscular and intradermal) is sufficient to elicit protection against both systemic and mucosal ricin challenges.     

Protective immunity to ricin toxin conferred by antibodies against the toxin's binding subunit (RTB)

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5×LD₅₀s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG₁ antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5×LD₅₀ dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB's carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin.     

Combination of two candidate subunit vaccine antigens elicits protective immunity to ricin and anthrax toxin in mice

In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population.     

Anthrax vaccine recipients lack antibody against the loop neutralizing determinant: A protective neutralizing epitope from Bacillus anthracis protective antigen

BackgroundEpitope-focused immunogens can elicit antibody against the loop neutralizing determinant (LND), a neutralizing epitope found within the 2β2-2β3 loop of protective antigen (PA), which can protect rabbits from high-dose inhalation challenge with Bacillus anthracis Ames strain. Interestingly, data suggests that this epitope is relatively immunosilent in rabbits and non-human primates immunized with full length PA.MethodsTo determine whether the LND is immunosilent among humans vaccinated with PA, we screened antisera from AVA- or placebo-vaccinees from a clinical trial for antibody reactive with the LND.ResultsAVA-vaccinee sera had significant PA-specific antibody compared to placebo-vaccinee sera; however, sera from the two cohorts were indistinguishable with regard to the frequency of individuals with antibody specific for the LND.ConclusionsAVA-vaccinees have a low frequency of antibody reactive with the LND. As with rabbits and non-human primates, the elicitation of LND-specific antibody in humans appears to require immunization with an epitope-focused vaccine.     

Endpoint and epitope-specific antibody responses as correlates of vaccine-mediated protection of mice against ricin toxin

The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD₅₀ of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration’s “Animal Rule.”     

C群脑膜炎奈瑟菌血清杀菌力试验的优化及其应用

目的 优化血清杀菌力试验方法,检测和分析A+C群脑膜炎球菌多糖疫苗接种前后人群血清对C群脑膜炎奈瑟菌(Nm)的杀菌抗体水平.方法 以C群Nm疫苗株(C11)和中国目前C群Nm流行株(053442)作为靶菌,国家Nm标准品测试血清为参考血清,选用Pel-Freez幼兔补体,确定靶菌最适工作浓度.122人接种A+C群脑膜炎球菌多糖疫苗前后分别采集血清标本,共244份,进行C群Nm杀菌抗体水平的检测.结果 菌株C11和053442均可作为靶菌用于血清杀菌力试验;靶菌的工作浓度为A 0.35(600 nm)的菌液稀释4×104倍;A+C群脑膜炎球菌多糖疫苗接种前,122份血清对C11和053442的杀菌抗体几何平均滴度(GMT)分别为1:1.75和1:2.63,人群抗体保护率分别为9.8%和17.2%,A+C群脑膜炎球菌多糖疫苗接种后,122份血清的杀菌抗体GMT和人群保护率均显著升高(P<0.01),对疫苗株和流行株的杀菌抗体GMT分别为1:483.73和1:412.57,保护率分别为100%和95.9%.结论 接种A+C群脑膜炎球菌多糖疫苗,能够显著提高人群对不同亚型的C群Nm的抵抗力,但仍需要针对不同的靶菌进行疫苗免疫效果的监测.     

Amplification of immune responses against a DNA-delivered idiotypic lymphoma antigen by fusion to the B subunit of E. coli heat labile toxin

The pentameric B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) is not only highly immunogenic itself, but can also act as a potent adjuvant or carrier to increase immune responses to other antigens. In this study, we investigated the ability of EtxB to promote anti-tumor immune responses using a fusion DNA vaccine design. EtxB was genetically linked to a single chain Fv sequence derived from the idiotypic immunoglobulin antigen (Id) of the mouse BCL1 B-cell lymphoma. We found that the EtxB–BCL1scFv fusion protein with a specifically selected linker retained the ability to pentamerize and to bind the GM1 ganglioside. Immunization of mice with the pEtxB–BCL1scFv DNA construct generated high levels of Id-specific antibody and protected against lethal tumor challenge. The immuno-enhancing activities of EtxB were highly dependent on GM1 binding, since fusion constructs unable to pentamerize or to bind to GM1 were less effective. Thus, the EtxB fusion vaccine approach may be an attractive strategy to increase the potency of tumor vaccines.     

Display of neutralizing epitopes of Canine parvovirus and a T-cell epitope of the fusion protein of Canine distemper virus on chimeric tymovirus-like particles and its use as a vaccine candidate both against Canine parvo and Canine distemper

Expression of Physalis mottle tymovirus coat protein in Escherichia coli was earlier shown to self-assemble into empty capsids that were nearly identical to the capsids formed in vivo. Amino acid substitutions were made at the N-terminus of wild-type Physalis mottle virus coat protein with neutralizing epitopes of Canine parvovirus containing the antigenic sites 1–2, 4 and 6–7 and T-cell epitope of the fusion protein of Canine distemper virus in various combinations to yield PhMV1, PhMV2, PhMV3, PhMV4 and PhMV5. These constructs were cloned and expressed in E. coli. The chimeric proteins self-assembled into chimeric tymovirus-like particles (TVLPs) as determined by electron microscopy. The TVLPs were purified by ultracentrifugation and injected into guinea pigs and dogs to determine their immunogenicity. Initial immunogenicity studies in guinea pigs indicated that PhMV3 gave a higher response in comparison to the other TVLPs for both CPV and CDV and hence all further experiments in dogs were done with PhMV3. HI was done against different isolates obtained from various parts of the country. Protective titres indicated the broad spectrum of the vaccine. In conclusion the study indicated that the above chimeric VLP based vaccine could be used in dogs to generate a protective immune response against diseases caused by both Canine parvo and Canine distemper virus.     

Immunogenic display of diverse peptides,including a broadly cross-type neutralizing human papillomavirus L2 epitope,on virus-like particles of the RNA bacteriophage PP7

The immunogenicity of an antigen can be dramatically increased by displaying it in a dense, multivalent context, such as on the surface of a virus or virus-like particle (VLP). Here we describe a highly versatile VLP platform for peptide display based on VLPs of the RNA bacteriophage PP7. We show that this platform can be used for the engineered display of specific peptide sequences as well as for the construction of random peptide libraries. Peptides representing the FLAG epitope, the V3 loop of HIV gp120, and a broadly cross-type neutralizing epitope from L2, the minor capsid protein of Human Papillomavirus type 16 (HPV16), were inserted into an exposed surface loop of a form of PP7 coat protein in which the two identical polypeptides of coat were fused together to form a single-chain dimer. The recombinant proteins assembled into VLPs, displayed these peptides on their surfaces, and induced high-titer antibody responses. The single-chain dimer was also highly tolerant of random 6-, 8-, and 10-amino acid insertions. PP7 VLPs displaying the HPV16 L2 epitope generated robust anti-HPV16 L2 serum antibodies after intramuscular injection that protected mice from genital infection with HPV16 pseudovirus as well as a heterologous HPV pseudovirus type, HPV45. Thus, PP7 VLPs are well-suited for the display of a wide diversity of peptides in a highly immunogenic format.     

建德市肾综合征出血热Ⅰ型灭活疫苗随机对照现场试验——中期免疫学效果及流行病学效果评价

对建德市ＨＦＲＳⅠ型灭活疫苗随机对照现场随访３９个月,采集免前和免后双份血清４０１人,检测ＩＦＡＴ和ＭＣＰＥＮＴ。免前ＩＦＡ抗体阴性者３６９人,免后ＩＦＡ阳性率和几何平均滴度分别是１５．９９％和１．６６,免后ＭＣＰＥＮＴ阳性率和几何平均滴度分别是７．３２％和１．１２;免前ＩＦＡ抗体阳性者３２人,免后ＩＦＡ阳性率和几何平均滴度分别是６８．７５％和１５．３５;疫苗保护发病效果为１００％,且对Ⅱ型病例也有保护作用。疫苗的中期保护发病效果较好,免后３９个月抗体水平下降幅度很大。提示疫苗的免疫原性评价应以基础和加强接种后２～３周的抗体水平为主要指标。     

A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev.1 vaccination

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably due to its decameric arrangement and remarkable stability. In this work we decided to develop a chimera with the scaffold protein BLS decorated with 10 copies of a known protective epitope derived from an outer membrane protein of 31kDa (Omp31) from Brucella spp. Vaccination of BALB/c mice with the chimera as a recombinant protein (rBLSOmp31) provided the best protection level against Brucella ovis, which was higher than the given by the co-delivery of both recombinant proteins (rBLS + rOmp31) and similar than the control vaccine Brucella melitensis strain Rev.1. Moreover rBLSOmp31 induced protection against Brucella melitensis but to a lesser degree than Rev.1. The chimera induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific T helper 1 and cytotoxic T responses. In conclusion, our results indicate that BLSOmp31 could be a useful candidate for the development of subunit vaccines against brucellosis since it elicits humoral, T helper and cytotoxic immune responses and protection against smooth and rough species of Brucella.     

Effect of mate tea (Ilex paraguariensis) on the expression of the leukocyte NADPH oxidase subunit p47phox and on circulating inflammatory cytokines in healthy men: a pilot study

Increased superoxide production by phagocytic NADPH oxidase has been associated with inflammatory conditions. Growing evidences suggest that dietary polyphenols may modulate the expression of NADPH oxidase subunits. Herein, we examined whether soluble mate tea (SMT) consumption – a polyphenol-rich beverage – affects the expression of the leukocyte NADPH oxidase protein p47^phox and/or circulating biomarkers of inflammation and antioxidant biomarkers in humans. In a two-phase study, nine men were requested to drink water (control) for 8 d and then follow a second 8-d period drinking SMT. Blood samples were analysed for p47^phox protein in CD16⁺/CD14^? cells, interleukin (IL)-1β (IL-1β), tumour necrosis factor-alpha (TNF-α), IL-6, total phenols, and reduced and oxidised glutathione (GSH and GSSG, respectively) after each study phase. After SMT intake, CD16⁺/CD14^? cells' p47^phox protein and serum TNF-α and IL-6 levels were significantly attenuated (P?<?.05) while plasma phenolic compounds and blood GSH:GSSG ratio were significantly enhanced (P?<?.05). Consumption of SMT favourably affected leukocytes' p47^phox expression and inflammatory cytokine and antioxidants levels in peripheral blood, which may help decrease oxidative stress and low-grade inflammation.     

百草枯对A549细胞中解整合素-金属蛋白酶17表达的影响

目的构建体外百草枯（paraquat,PQ）细胞纤维化模型,观察PQ对A549细胞中解整合素-金属蛋白酶17（ADAM17）表达的影响,探讨ADAM17在PQ中毒致肺纤维化中的作用。方法体外培养A549细胞,分为正常对照组、不同浓度PQ组,应用CCK-8检测细胞活力,筛选PQ浓度和时间,显微镜下观察细胞形态,ELISA测定各组纤维化标志物I型胶原（type I collagen,Col I）和纤连蛋白（fibronectin,FN）的表达,建立细胞纤维化模型;免疫细胞化学检测A549细胞中ADAM17的分布情况,RT-PCR和Western blot分别半定量检测ADAM17 mRNA及蛋白水平的表达情况。结果（1）随着PQ浓度增加及作用时间延长,A549细胞活力呈下降趋势,差异有统计学意义（P〈0.05）,呈剂量依赖性和时间依赖性。（2）正常的A549细胞融合呈铺路石样生长,排列比较紧密,经PQ诱导后细胞排列较松散,细胞间连接变疏松,部分细胞溶解、死亡。（3）ELISA显示,随PQ浓度增加,Col I和FN表达增强,差异有统计学意义（P〈0.05）;随PQ时间延长,Col I和FN表达也逐渐增强,差异有统计学意义（P〈0.05）,成功建立PQ细胞纤维化模型。（4）免疫细胞化学显示,ADAM17在A549细胞胞浆表达。（5）RT-PCR和Western blot表明,随着PQ浓度增加,ADAM17 mRNA及蛋白水平表达明显增加,差异有统计学意义（P〈0.05）,以PQ 200 umol/L时最为明显。随着PQ作用时间延长,ADAM17 mRNA及蛋白表达水平也明显增加,差异有统计学意义（P〈0.05）,在24 h达到高峰。结论百草枯可引起肺泡上皮细胞形态学改变,导致细胞损伤,成功建立细胞的纤维化模型,对A549细胞的毒性作用具有剂量和时间依赖性。ADAM17在PQ诱导的A549细胞中过表达,可能参与了百草枯诱导的肺纤维化过程。     

A serological test for leprosy based on competitive inhibition of monoclonal antibody binding to the MY2a determinant of Mycobacterium leprae

A novel serological assay for leprosy has been devised on the basis of serum inhibition of binding of ¹²⁵I-labelled MLO4 monoclonal antibody to Mycobacterium leprae sonicate-coated microtitre plates. Antibodies were detected in 93% of lepromatous leprosy patients, whereas controls from the endemic area, including leprosy contacts and patients with tuberculosis, were serologically negative. The specificity and efficacy of the test may offer an advantage over previously used techniques.     

Glutamine as a modulator of the immune system of critical care patients: effect on Toll-like receptor expression. A preliminary study

OBJECTIVE: We evaluated the expression of Toll-like receptors 2 and 4 (TLR-2 and TLR-4) in circulating monocytes from peripheral blood of critical care patients treated with and without glutamine. Because no research has been published to date on the effect of glutamine on TLR receptors in critical patients, it was determined in an initial sample of 30 patients. METHODS: This was a prospective, randomized, single-blind study with 15 patients assigned to receive parenteral nutrition with a daily glutamine supplement of 0.35 g/kg. The control group received isocaloric-isonitrogenous parenteral nutrition. Blood samples were extracted before beginning the treatment and at 5 and 14 d. Expressions of CD14, TLR-2, and TLR-4 were determined by flow cytometry. Levels of TLRs were expressed as mean fluorescence intensity (mfi). RESULTS: Basal characteristics were similar in both groups. The expressions of TLR-2 in the treatment group with glutamine were 4.67 +/- 3.82 mfi before treatment, 3.91 +/- 2.04 mfi at 5 d, and 4.28 +/- 2.47 mfi at 14 d. The expressions of TLR-2 in the control group were 5.49 +/- 3.20 mfi before treatment, 4.48 +/- 2.15 mfi at 5 d, and 4.36 +/- 2.36 mfi at 14 d. The expressions of TLR-4 in the treatment group were 1.65 +/- 1.89 mfi before treatment, 1.23 +/- 1.10 mfi at 5 d, and 1.77 +/- 1.97 at 14 d. The expressions of TLR-4 in the control group were 1.51 +/- 1.76 mfi before treatment, 1.36 +/- 0.99 mfi at 5 d, and 1.26 +/- 0.59 mfi at 14 d. Infections were detected in 11 patients who received glutamine and 13 control patients (P = 0.51). CONCLUSION: In critical care patients, parenteral nutrition supplemented with glutamine does not increase the expression of TLR-2 or TLR-4 in peripheral blood monocytes.     

Effectiveness of an Intervention Programme on Adherence to the Mediterranean Diet in a Preschool Child: A Randomised Controlled Trial

Background: The Mediterranean diet is considered one of the dietary patterns with the most accumulated scientific evidence on health benefits. In children, it has positive effects in the prevention of obesity and cardiovascular diseases, as well as in the prevention of diabetes. We aimed to evaluate the medium-term efficacy of an intervention programme, targeting adherence to the Mediterranean diet among preschool children. Methods: In a randomised, parallel trial of participants aged 3–5 years, a school garden was attended in the experimental group, and in the control group, the usual content on the human body and health were taught. Adherence to the Mediterranean diet was assessed using the KIDMED questionnaire, controlling for weight, height, body mass index (BMI) and socio-demographic variables. Results: A reduction in BMI was found in the experimental group after one year and at the end of the follow-up period. In the overall score obtained in the KIDMED survey, a statistical trend was found between the two groups (p = 0.076). In multivariate analysis, consumption of pulses more than once a week’ was predictive of improved diet quality, with an Odds Ratio (OR) in the experimental group of 1.382 (95% CI 1.126–1.695; p = 0.009). Conclusions: The experimental approach improved the quality of the participants’ diet, increasing adherence to the Mediterranean diet due to increased consumption of plant-based protein.