Role of antigen valency and pattern recognition receptor ligands in HPV vaccine-induced durable B cell memory

摘要 大多数疫苗通过产生抗体来保护人体。减毒活疫苗可诱导持久的体液免疫； 然而，许多亚单位疫苗的体液免疫力随着时间的推移而减弱。因为减毒活疫苗可能 并不是所有的病原体或接受者都可行，亚基疫苗的开发可以诱导有效和长期的- 活的抗体反应是必不可少的。几十年来，疫苗学一直专注于开发亚单位疫苗， 引起对人类病原体的强健和/或广泛的抗体反应。然而，对公众健康至关重要的是 亚单位疫苗还能产生持久的抗体反应。因此，需要进行基础研究，以了解如何 可靠地设计这样的疫苗。高效人乳头瘤病毒9型亚单位疫苗(HPV-9) 预防九种通常致癌的HPV类型(例如，HPV16)。此外，它的抗体持久性 优于许多其他已获批准的亚单位疫苗。因此，HPV-9代表了一种模型亚单位疫苗 用于研究产生强健和持久的抗体反应。与大多数其他亚单位疫苗不同，HPV 疫苗由一种高度重复、高价的抗原组成：病毒样颗粒(VLP)。每个VLP HPV TYPE由360个单位的主要衣壳蛋白L1组装而成。此外，L1结合DNA，HPV-9包含 重组L1 DNA。无论是高抗原价还是抗原相关DNA通常都会增强 持久B细胞记忆的形成还没有得到很好的研究。在HPV方面，L1 VLP可诱导更高的峰中和 相对于L1五聚体的抗体反应和抗VLP抗体反应依赖于MyD88，它是 DNA传感途径。目前缺乏的是对疫苗DNA是否会增强病毒数量的了解 对HPV-9的抗体反应以及抗原效价或疫苗DNA在诱导HPV-9感染中的作用 经久耐用的HPV特异性B细胞存储器。这项提案将通过两个目标解决这些差距。第一个目标是 测试高峰期和长寿的HPV-9诱导的B细胞反应对DNA传感器的依赖性。野生型小鼠和 不同DNA传感途径缺陷的小鼠将被HPV-9或明矾免疫。之前和在不同的 每次接种HPV-9后的时间点、HPV特异性中和抗体、记忆B细胞和骨髓血浆 我们将分析细胞的反应。通过这种方式，DNA传感在刺激高峰期、回忆和长期中的作用 (≥12个月)将评估B细胞反应。第二个目标是评估长寿患者的依赖性 HPV特异性B细胞记忆对抗原效价的影响。将用三种剂量的HPV16单体免疫小鼠 L1(1-MERS)、HPV 16 L1五聚体(5-MERS)或HPV 16 L1 VLP(360-MERS)。在前后的不同时间 每个抗原剂量、HPV16特异性中和抗体、生发中心B细胞、记忆B细胞和骨 将分析骨髓浆细胞的反应。对1聚体、5聚体和360聚体抗原的反应将是 比较一下。通过这种方式，将测试抗原价在诱导长期B细胞记忆中的作用。这 这项工作将促进对HPV-9如何实现持久抗体反应的了解，并为这一发展提供信息 更持久的体液免疫和更好的对其他病原体的保护。

ABSTRACT Most vaccines protect by generating antibodies. Live attenuated vaccines induce lasting humoral immunity; however, humoral immunity for many subunit vaccines wanes over time. Because live attenuated vaccines may not be feasible for all pathogens or recipients, the development of subunit vaccines that elicit potent and long- lived antibody responses is essential. Vaccinology has focused for decades on developing subunit vaccines that elicit robust and/or broad antibody responses to human pathogens. However, it is crucial to public health that subunit vaccines also generate durable antibody responses. Thus, basic research is needed to know how to reliably design such vaccines. The highly effective 9-type human papillomavirus subunit vaccine (HPV-9) protects against nine HPV types that commonly cause cancer (e.g., HPV 16). Moreover, its antibody durability is superior to that of many other approved subunit vaccines. Thus, HPV-9 represents a model subunit vaccine for studying the generation of robust and enduring antibody responses. Unlike most other subunit vaccines, HPV vaccines are comprised of a highly repetitive, high valency antigen: virus like particles (VLP). Each VLP HPV type assembles from 360 units of its major capsid protein, L1. In addition, L1 binds DNA, and HPV-9 contains recombinant L1 DNA. Whether high antigen valency or antigen-associated DNA generally enhances the formation of durable B cell memory is not well studied. With regard to HPV, L1 VLPs elicit higher peak neutralizing antibody responses than L1 pentamers and anti-VLP antibody responses depend on MyD88, a component of a DNA sensing pathway. What is lacking is an understanding of whether vaccine DNA enhances the magnitude of antibody responses to HPV-9 and whether antigen valency or vaccine DNA plays a role in the induction of durable HPV-specific B cell memory. This proposal will address those gaps through two aims. The first aim is to test the dependency of peak and long-lived HPV-9-elicited B cell responses on DNA sensors. Wildtype mice and mice deficient in various DNA sensing pathways will be immunized with HPV-9 or alum. Before and at various time points after each HPV-9 dose, HPV-specific neutralizing antibody, memory B cell, and bone marrow plasma cell responses will be analyzed. In this way, the role of DNA sensing in stimulating peak, recall, and long-term (≥12 months) B cell responses will be assessed. The second aim is to evaluate the dependency of long-lived HPV-specific B cell memory on antigen valency. Mice will be immunized with three doses of monomeric HPV 16 L1 (1-mers), HPV 16 L1 pentamers (5-mers), or HPV 16 L1 VLPs (360-mers). Before and at various times after each antigen dose, HPV 16-specific neutralizing antibody, germinal center B cell, memory B cell, and bone marrow plasma cell responses will be analyzed. Responses to 1-mer, 5-mer, and 360-mer antigens will be compared. In this way, the role of antigen valency in the induction of long-term B cell memory will be tested. This work will advance knowledge of how HPV-9 achieves durable antibody responses and inform the development of vaccines with longer-lasting humoral immunity and improved protection against other pathogens.