Falk Jessica J, Winkelmann Martina, Schrezenmeier Hubert, Stöhr Dagmar, Sinzger Christian, Lotfi Ramin
Institute for Virology, University Hospital Ulm, Ulm.
University Hospital Ulm.
Transfusion. 2017 Feb;57(2):412-422. doi: 10.1111/trf.13906. Epub 2016 Nov 10.
Hyperimmunoglobulins are frequently applied for prophylaxis and treatment of human cytomegalovirus (HCMV) infections but were only marginally effective in meta-analyses of clinical studies. This might be partially due to selection of donors rather for total anti-HCMV titers than for neutralizing capacities. To improve efficacy against HCMV infection, we aimed at developing a high-throughput screening method for identification of blood donors with highly and broadly neutralizing capacities.
Using a Gaussia luciferase-expressing reporter virus, 1000 HCMV immunoglobulin (Ig)G-positive plasma samples with known anti-HCMV immunoglobulin titers were analyzed regarding their neutralization titers against fibroblast and endothelial cell infection. Based on these results, a high-throughput screening was designed. Highly neutralizing plasma samples were further tested 1) by an enzyme-linked immunosorbent assay-based neutralization assay regarding efficiency against different HCMV strains and 2) for their efficiency compared to commercially available hyperimmunoglobulins.
Total anti-HCMV immunoglobulin titers did not correlate with neutralization. Mean neutralization capacities were 15-fold higher in endothelial cells compared to fibroblasts. All plasma samples neutralizing fibroblast infection were at least equally effective against infection of endothelial cells, providing the possibility to simplify our screening method by testing only fibroblasts as target cells with a plasma dilution of 1 in 400. Of the nine tested top HCMV neutralizers, four were broadly effective against different HCMV strains. All nine were significantly superior to hyperimmunoglobulins.
Donors with highly and broadly neutralizing capacities can be identified by a two-step high-throughput screening approach. This may provide a basis for improved antibody-based treatment or prophylaxis of HCMV infections.
高免疫球蛋白常用于预防和治疗人类巨细胞病毒（HCMV）感染，但在临床研究的荟萃分析中效果甚微。这可能部分归因于供体的选择是基于总抗HCMV滴度而非中和能力。为提高抗HCMV感染的疗效，我们旨在开发一种高通量筛选方法，以鉴定具有高中和能力和广泛中和能力的献血者。
使用表达高斯荧光素酶的报告病毒，分析了1000份已知抗HCMV免疫球蛋白滴度的HCMV免疫球蛋白（Ig）G阳性血浆样本对成纤维细胞和内皮细胞感染的中和滴度。基于这些结果，设计了高通量筛选。对高中和活性的血浆样本进一步进行如下测试：1）通过基于酶联免疫吸附测定的中和试验，检测其对不同HCMV毒株的有效性；2）与市售高免疫球蛋白相比，检测其有效性。
总抗HCMV免疫球蛋白滴度与中和作用不相关。内皮细胞中的平均中和能力比成纤维细胞高15倍。所有中和成纤维细胞感染的血浆样本对内皮细胞感染至少同样有效，这使得我们有可能通过仅以1:400的血浆稀释度测试成纤维细胞作为靶细胞来简化筛选方法。在测试的9种顶级HCMV中和剂中，有4种对不同的HCMV毒株具有广泛的有效性。所有9种均显著优于高免疫球蛋白。
通过两步高通量筛选方法可以鉴定出具有高中和能力和广泛中和能力的供体。这可能为改进基于抗体的HCMV感染治疗或预防提供基础。
