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。宿主对抗这些不断变化的表位的一个极好的方法是培养广泛中和的抗体。这些抗体识别表面蛋白的保守功能区域，从而提供针对特定病原体的长期保护。虽然人类可以产生针对疟疾等慢性感染的广泛中和抗体，但这通常需要5年以上的时间，并且仅在5-10%的慢性感染人群中明显存在。另一方面，牛在6周内产生广泛中和的抗体，以对抗高度逃避的病原体，如艾滋病毒。这个项目旨在了解分子机制，通过牛产生广泛中和抗体如此有效。这具有重大的健康和财富意义：通过利用牛使用的机制，这一途径可以在其他生物体中得到增强，从而能够产生用于治疗目的的人源化广泛中和抗体。此外，它还可以为研究目的开发更可靠的抗体。广泛中和抗体基因的一个共同特征是将一段DNA精确地插入可变的外显子。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域。在人类中，这些插入的序列似乎起源于转录区，但在牛中的起源以及插入的机制仍然未知。在序列插入之前，牛必须接触抗原。在插入之后，这些序列会发生广泛的突变，从而增加抗体的多样性。因此，为了了解产生广泛中和抗体的分子机制，该项目将首先使用克隆、下一代测序和生物信息学技术的组合来确定插入抗体基因的序列的起源。接下来，它将检查插入抗体基因发生的细胞类型。抗体基因在骨髓前b细胞中通过V(D)J重组产生，并在淋巴结生发中心进一步修饰。将使用现有的牛抗体和流式细胞术以及分子克隆技术研究插入发生的细胞类型。第三，深入研究DNA插入的机制。一个受欢迎的假设是，RNA被插入DNA断裂，然后在DNA修复过程中进行逆转录。这一机制将使用现有的牛B细胞系进行广泛的研究，然后在原代细胞中进行验证。具体来说，相关酶的表达将使用qPCR进行检测，外源添加的rna插入和逆转录的能力将使用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如何发生，以进一步使这些广泛中和的抗体多样化。具体来说，它将询问AID如何如此容易地靶向具有长CDR3的抗体基因。总之，这些研究将为在小鼠细胞中产生人源化的广泛中和抗体奠定基础，这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.