分子定义结核菌素配方的优化:重组融合蛋白和表位手术。

IF 5.4 2区 医学 Q1 MICROBIOLOGY
Sonya Middleton, Mahavir Singh, Michael Coad, Si Palmer, Tom Holder, Sabine Steinbach, Rebecca Hardiman, H Martin Vordermeier, Gareth J Jones
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引用次数: 0

摘要

牛结核病是一种具有全球经济和人畜共患病重要性的家畜传染病。监测方案在很大程度上依赖于使用结核菌素纯化蛋白衍生物(PPDs)的皮肤试验。ppd在生产、表征和性能方面存在许多限制。最近,我们开发了一种分子定义结核菌素(MDT),克服了这些局限性。MDT配方包括8种抗原,最初表现为7种重组蛋白(ESAT6、CFP10、Rv3615c、Rv3020c、Rv1789、Rv3478和Rv3810)和一个20合成的肽库,代表Rv3616c;重组Rv3616c无法产生。在本文中,我们描述了简化MDT公式所采取的步骤。首先,将8个蛋白中的7个配制成3个重组融合蛋白。其次,利用重叠肽探测感染牛的t细胞,鉴定Rv3616c的非免疫原性区域。设计了一种新的变体Rv3616c-S4,然后将其配制为(i)作为MDT-S4制剂中的独立蛋白,(ii)与Rv1789融合用于MDT-F制剂。这些新型MDT制剂在实验感染动物中具有与PPD-B相当的信号强度。仅包含融合蛋白的MDT-F在相对敏感性(> 2mm; 96%, 95% CI 80-100, n = 24)和特异性(> 2mm; 100%, 95% CI 89%-100%, n = 30)方面的表现与比较宫颈结核菌素皮肤试验一样好。总之,我们已经开发出一种新颖的、简化的MDT配方,这大大推进了我们在许可方面的努力,并有可能取代20世纪30年代开发的ppd。重要性:牛结核病是一种具有全球经济和人畜共患病重要性的家畜传染病。监测项目很大程度上依赖于使用结核菌素的皮肤试验。这些是活细菌培养物的粗蛋白质提取物,在其表征、标准化、生产和性能方面受到许多限制。我们的目标是开发一种皮肤试验试剂,由八种确定的抗原组成,可以取代结核菌素。本研究描述了这种“分子定义结核菌素”(MDT)试剂的改进,使其成为包含所有八种抗原的融合蛋白配方。MDT定义明确,易于标准化,并在实验感染、未感染和对约翰氏病敏感的牛中提供良好的测试性能。融合蛋白配方是迈向注册和上市产品的重要一步。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Optimization of a molecularly defined tuberculin formulation: recombinant fusion proteins and epitope surgery.

Bovine tuberculosis is an infectious livestock disease of global economic and zoonotic importance. Surveillance programs are largely dependent on the skin test that utilizes purified protein derivatives (PPDs) of tuberculin. PPDs suffer from a number of limitations regarding their production, characterization, and performance. Recently, we developed a molecularly defined tuberculin (MDT) that overcame these limitations. The MDT formulation comprises eight antigens, initially presented as seven recombinant proteins (ESAT6, CFP10, Rv3615c, Rv3020c, Rv1789, Rv3478, and Rv3810) and one 20-synthetic peptide pool representing Rv3616c; the recombinant Rv3616c could not be produced. In this paper, we describe the steps taken to simplify the MDT formulation. First, seven of the eight proteins were formulated as three recombinant fusion proteins. Second, the non-immunogenic regions of Rv3616c were identified, using overlapping peptides to probe T-cells from infected cattle. A novel variant, Rv3616c-S4, was designed and then formulated (i) as a stand-alone protein in the MDT-S4 formulation and (ii) fused to Rv1789 for use in the MDT-F formulation. These novel MDT formulations gave comparable signal strength to PPD-B in experimentally infected animals. The MDT-F, comprising only fusion proteins, performed as well as the comparative cervical tuberculin skin test, in terms of relative sensitivity (>2 mm; 96%, 95% CI 80-100, n = 24) and specificity (>2 mm; 100%, 95% CI 89%-100%, n = 30). In conclusion, we have developed a novel, simplified MDT formulation that has significantly advanced our efforts toward licensure and which has the potential to replace the PPDs developed in the 1930s.

Importance: Bovine tuberculosis is an infectious livestock disease of global economic and zoonotic importance. Surveillance programs are largely dependent on the skin test, which utilizes tuberculins. These are crude protein extracts of live bacterial cultures, which suffer from a number of limitations regarding their characterization, standardization, production, and performance. We aim to develop a skin test reagent composed of eight defined antigens that could replace the tuberculins. This study describes the refinement of this "molecularly defined tuberculin" (MDT) reagent into a fusion protein formulation comprising all eight antigens. The MDT is well defined, easily standardized, and delivers good test performance in experimentally infected, non-infected cattle and cattle sensitized to Johne's disease. The fusion protein formulation is an important step on the developmental pathway to a registered and marketable product.

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来源期刊
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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