Leukocyte Signaling in the Elderly and Vaccine Immunogenicity

描述(申请人提供)：流感病毒感染继续在老年人(65岁或以上)中造成不成比例的死亡，尽管这一人群每年接种灭活三价流感疫苗(TIV)的水平很高。总体而言，TIV在老年人中的有效性和免疫原性显著低于年轻人，尽管由于尚不清楚的原因存在很大的个体差异。TIV含有血凝素(HA)和神经氨酸酶(NA)主要的病毒表面蛋白，没有佐剂，主要通过诱导针对HA的中和抗体来提供保护。TIV诱导的流感特异性CD4T细胞反应可能有助于抗体反应，并与CD8T细胞反应一起，在接种疫苗后发生感染时增强病毒清除。目前尚不清楚加入强效适应性免疫佐剂，如阳离子脂/DNA复合体(CLDC)能在多大程度上提高老年人对TIV和其他疫苗的免疫原性。Juvaris BioTreateutics公司赞助的一项第二阶段人体研究正在评估单次肌肉注射TIV/CLDC对472名老年男性和女性的最佳流感特异性体液和T细胞反应的安全性、有效性和剂量需求。作为这项研究的一部分，GLP认证的流感特异性抗体以及接种后基线和多个时间点的CD4和CD8 T细胞反应的分析正在进行中。这些免疫原性结果的可用性，以及多份冷冻保存的PBMC，为在该项目中确定老年人疫苗免疫原性的预测因素提供了一个独特的机会。中心假说是，一些老年人对TIV的体液免疫原性和T细胞免疫原性受损反映了抗原提呈细胞(APC)、B细胞和T细胞信号的内在局限性，而这些局限性反过来又是microRNAs(MiRNAs)改变mRNA表达的结果。这一假设将通过确定这些老年受试者在TIV或TIV/CLDC接种后的免疫原性是否可以通过他们的单核细胞、B细胞和T细胞在细胞因子受体(如IL-21)、先天免疫配体(如CpG DNA)和抗原(即B细胞和T细胞受体)结合后的信号能力来预测，这一改进的磷酸盐流式细胞仪分析方法进行了评估。同时，单核细胞、B细胞以及CD4和CD8T细胞的主要亚群的频率将通过多色流式细胞术来测定，这在解释磷酸盐流动结果中将是重要的。然后，将评估作为TIV或TIV/CLDC免疫原性最可靠预测指标的白细胞亚群和刺激，以评估其基础和刺激依赖的mRNA和miRNA表达水平，重点关注已知或可能影响信号转导的变化。总之，这些研究将确定白细胞亚群信号水平在老年人对TIV和TIV/CLDC产生适应性免疫反应中的预测价值和生物学重要性。他们还将提供关于特定mRNAs和miRNAs在调节信号和激活诱导的涉及疫苗免疫原性的基因表达方面所起作用的新的机械性见解。 公共卫生相关性(由申请人提供)：在老年人等高危人群中使用疫苗预防流感等感染的一个主要限制是，很难预测免疫接种是否会有效地增强免疫反应，以保护个人免受传染病的侵袭。这项研究将确定免疫系统白细胞功能的测试，这些测试可以用来预测为老年人接种流感疫苗是否可能充分刺激免疫系统，为该人提供免受感染的保护。这些测试将有助于更有效地使用现有疫苗，还将有助于开发一种使用佐剂的新的、更有效的流感疫苗，佐剂是一种添加到疫苗中以增强免疫反应的物质。

DESCRIPTION (provided by applicant): Influenza viral infection continues to cause disproportionate mortality in the elderly (those 65 years of age or older) despite a high level of annual immunization of this population with inactivated trivalent influenza vaccine (TIV). The efficacy and immunogenicity of TIV in the elderly overall is substantially lower than in younger adults, although there is substantial individual variation for reasons that are not well understood. TIV, which contains the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins and is unadjuvanted, provides protection mainly by inducing neutralizing antibodies to HA. Influenza-specific CD4 T-cell responses induced by TIV may contribute to the antibody response and, in conjunction with CD8 T-cell responses, may enhance viral clearance in the event that infection occurs despite vaccination. It is unclear to what extent the inclusion of potent adjuvants for adaptive immunity, such as cationic lipid/DNA complexes (CLDC) could boost immunogenicity in the elderly to TIV and other vaccines. A phase II human study sponsored by Juvaris Biotherapeutics, Inc., is evaluating the safety, efficacy and dose requirement of a single intramuscular dose of TIV/CLDC for optimal influenza-specific humoral and T-cell responses in 472 elderly men and women. As part of this study, GLP-certified assays of influenza-specific antibody and CD4 and CD8 T-cell responses at baseline and at multiple time points post-vaccination are in progress. The availability of these immunogenicity results, along with multiple aliquots of cryopreserved PBMCs, provides a unique opportunity to determine predictors of vaccine immunogenicity in the elderly in this project. The central hypothesis is that impaired humoral and T-cell immunogenicity to TIV in some of the elderly reflects intrinsic limitations in the signaling of antigen-presenting cells (APCs), B cells, and T cells, and that these limitations are, in turn, the result of alterations of mRNA expression by microRNAs (miRNAs). This hypothesis will be tested by determining if immunogenicity after TIV or TIV/CLDC vaccination of these elderly subjects can be predicted by the capacity of their monocytes, B cells, and T cells to signal after engagement of receptors for cytokines, e.g., IL-21, innate immune ligands, e.g., CpG DNA, and antigen, i.e., the B-cell and T-cell receptors, as assessed by an improved phospho-flow cytometric assay. In parallel, the frequency of the major subsets of monocytes, B cells, and CD4 and CD8 T- cells will be determined by polychromatic flow cytometry, which will be important in the interpretation of the phospho-flow results. The leukocyte subsets and the stimuli that are most robust predictors of TIV or TIV/CLDC immunogenicity will then be evaluated for their basal and stimulus-dependent levels of mRNA and miRNA expression, focusing on changes that are known to or are likely to influence signaling. Together, these studies will define the predictive value and biological importance of the level of leukocyte subset signaling in generating adaptive immune responses to TIV and TIV/CLDC in the elderly. They will also provide new mechanistic insights as to the role that specific mRNAs and miRNAs play in regulating signaling and activation-induced gene expression involved in vaccine immunogenicity. PUBLIC HEALTH RELEVANCE (provided by applicant): A major limitation in using vaccines to prevent infections, such as influenza, in high-risk populations, such as the elderly, is that it is difficult to predict whether an immunization will be effective in sufficiently boosting the immune response to protect that individual from the infectious disease. This research will identify tests of the function of white blood cells of the immune system that can be used to predict whether vaccination of an elderly individual for influenza will be likely to sufficiently stimulate the immune system to provide that individual with protection from infection. These tests will help use current vaccines more effectively, and will also help in the development of a new, more potent vaccine for influenza that uses an adjuvant, a substance added to vaccines to boost the immune response.