Determining the mechanism by which highly effective, broadly neutralising antibodies are generated

病原体逃避宿主免疫系统的一个主要策略是频繁改变其表面蛋白上的表位。因此，每年都需要针对常见病毒的新疫苗，发生重大流行病的风险很大。对于宿主来说，对抗这些不断变化的表位的一个很好的方法是提高广泛的中和抗体。这些抗体识别表面蛋白的保守功能区，从而提供对特定病原体的长期保护。虽然人类可以产生广泛的中和抗体来对抗疟疾等慢性感染，但这通常需要五年以上的时间，只在5%-10%的慢性感染人群中表现明显。另一方面，牛在6周内就能产生针对高度可逃避的病原体(如艾滋病毒)的广泛中和抗体。该项目旨在了解牛如此高效地产生广谱中和抗体的分子机制。这具有重大的健康和财富影响：通过利用牛使用的机制，这一途径可以在其他生物中得到加强，从而能够产生人源化的广泛中和抗体，用于治疗目的。广谱中和抗体基因的一个共同特征是将一段DNA精确地插入可变外显子中。这增加了蛋白质的互补决定区3(CDR3)的长度，使抗体能够“到达”病原体蛋白质的保守区。在人类中，这些插入的序列似乎来自转录区域，但牛的来源以及插入的机制仍不清楚。在序列插入发生之前，牛必须接触抗原。插入后，序列发生广泛突变，以产生更多的抗体多样性。因此，为了了解产生广泛中和抗体的分子机制，该项目将首先结合克隆、下一代测序和生物信息学技术来确定插入抗体基因的序列的来源。接下来，它将检查发生抗体基因插入的细胞类型(S)。抗体基因通过V(D)J重组在骨髓中的原B细胞中产生，并在淋巴生发中心进一步修饰。将利用现有的牛抗体和流式细胞术，结合分子克隆技术，研究发生插入的细胞类型(S)。第三，深入探讨DNA插入的机制。一个受欢迎的假设是，RNA被插入到DNA断裂中，然后在DNA修复过程中被反转录。这一机制将使用现有的牛B细胞株进行广泛研究，然后在原代细胞中进行验证。具体地说，相关酶的表达将通过qPCR进行检测，外源添加的RNA插入和反转录的能力将通过细胞培养和分子生物学技术进行检测。最后，该项目将研究高水平的体细胞超突变是如何发生的，以进一步使这些广泛中和抗体多样化。具体地说，它将询问AID是如何如此容易地靶向具有长CDR3的抗体基因的。这些研究将为在小鼠细胞中生产人源化的广泛中和抗体奠定基础，这一抗体具有巨大的治疗潜力。

A major strategy used by pathogens to evade the host immune system is to frequently alter epitopes on their surface proteins. Consequently, new vaccines against common viruses are required every year and the risk of major pandemics is significant. An excellent way for hosts to combat these constantly changing epitopes is to raise broadly neutralising antibodies. These antibodies recognise conserved functional regions of surface proteins and thus offer long term protection against a given pathogen. Whilst humans can generate broadly neutralising antibodies against chronic infections such as malaria, this often takes more than five years and is only apparent in 5-10% of the chronically infected population. Cattle, on the other hand, generate broadly neutralising antibodies against highly evasive pathogens such as HIV within 6 weeks. This project aims to understand the molecular mechanism by which cattle generate broadly neutralising antibodies so efficiently. This has major health and wealth implications: By harnessing the mechanism used by cattle, this pathway could be enhanced in other organisms to enable the production of humanised broadly neutralising antibodies for therapeutic purposes. Moreover, it could allow development of more reliable antibodies for research purposes.A common feature of broadly neutralising antibody genes is the insertion of a piece of DNA precisely into the variable exon. This increases the length of complementarity determining region 3 (CDR3) of the protein, allowing the antibody to "reach" the conserved regions of pathogen proteins. In humans, these inserted sequences appear to originate from transcribed regions but the origin in cattle, as well as the mechanism of insertion, remains unknown. Cattle must be exposed to antigen before sequence insertion occurs. Following insertion, the sequences become extensively mutated to generate increased antibody diversity. Therefore, to understand the molecular mechanism by which broadly neutralising antibodies are produced, this project will firstly determine the origin of the sequences that are inserted into the antibody genes using a combination of cloning, next generation sequencing and bioinformatics techniques. Next, it will examine the cell type(s) in which insertion into the antibody gene occurs. Antibody genes are generated via V(D)J recombination in pro-B cells in the bone marrow and become further modified in germinal centres of lymph nodes. The cell type(s) in which inserts occur will be investigated using available bovine antibodies and flow cytometry, together with molecular cloning techniques.Thirdly, it will thoroughly investigate the mechanism of DNA insertion. A favoured hypothesis is that RNA is inserted into DNA breaks that is then reverse transcribed during DNA repair. This mechanism will be extensively investigated using available bovine B cell lines, followed by verification in primary cells. Specifically, expression of relevant enzymes will be examined using qPCR and the ability of exogenously added RNAs to be inserted and reverse transcribed will be examined using cell culture and molecular biology techniques.Finally, the project will investigate how the high level of somatic hypermutation occurs to further diversify these broadly neutralising antibodies. Specifically, it will ask how AID is targeted so readily to antibody genes with the long CDR3.Together, these studies will lay the groundwork for producing humanised broadly neutralising antibodies in mouse cells, which has immense therapeutic potential.