Liu Fangbing, Kumar Mukesh, Ma Qiangzhong, Duval Mark, Kuhrt David, Junghans Richard, Posner Marshall, Cavacini Lisa
Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
AIDS Res Hum Retroviruses. 2005 Oct;21(10):876-81. doi: 10.1089/aid.2005.21.876.
The F240 human monoclonal antibody specifically recognizes the disulfide loop-bonded immunodominant epitope of gp41 spanning residues 592-606 and expressed broadly on HIV-1 primary isolates. Despite broad reactivity with native virions and HIV-infected cells, the antibody fails to neutralize infection. However, cytoplasmic expression of single-chain antibody (scFv) directed against gp41 of HIV-1 provides a rationale means to inhibit the maturation of envelope protein. The variable regions of the heavy chain and light chain of human monoclonal antibody were amplified by PCR and linked by a 15 amino acid (GGSGS)3 linker in an orientation of VL-linker-VH and retroviral expression vectors were constructed to simultaneously express F240 scFv and eGFP to facilitate selection of scFv-producing cells. Incorporation of a human immunoglobulin signal sequence directed secretion of the F240 scFv (s-scFv) while an otherwise identical vector lacked this sequence (scFv) resulting in intracellular expression of scFv. Transduced human CD4+ H9 T cells were challenged with HIV. While both secreted and nonsecreted F240 scFv inhibited viral production, secretory F240 scFv was more potent. Thus, this novel approach to direct expression of a nonneutralizing scFv using the Ig signal sequence suggests that targeted therapy using antibodies to conserved, highly expressed epitopes may result in a decrease in viral production due to a reduction of viral assembly and/or transport and expression.
F240人源单克隆抗体特异性识别gp41中由二硫键环连接的免疫显性表位，该表位跨越592 - 606位氨基酸残基，在HIV-1原代分离株上广泛表达。尽管该抗体与天然病毒粒子和HIV感染细胞具有广泛反应性，但它无法中和感染。然而，针对HIV-1 gp41的单链抗体（scFv）的细胞质表达为抑制包膜蛋白成熟提供了一种合理的方法。通过PCR扩增人源单克隆抗体重链和轻链的可变区，并通过一个15个氨基酸（GGSGS）3的接头以VL-接头-VH的方向连接，构建逆转录病毒表达载体以同时表达F240 scFv和增强绿色荧光蛋白（eGFP），便于选择产生scFv的细胞。加入人免疫球蛋白信号序列可指导F240 scFv（s-scFv）的分泌，而一个除缺少该序列（scFv）外其他相同的载体则导致scFv在细胞内表达。用HIV攻击转导的人CD4 + H9 T细胞。虽然分泌型和非分泌型F240 scFv均能抑制病毒产生，但分泌型F240 scFv的效果更强。因此，这种利用Ig信号序列直接表达非中和性scFv的新方法表明，使用针对保守的、高表达表位的抗体进行靶向治疗可能会由于病毒组装和/或运输及表达的减少而导致病毒产生量下降。
