从人类免疫缺陷病毒1型感染者中筛选中和抗体的研究

Identification of neutralizing monoclonal antibodies against HIV-1 from virus infected individuals

目的利用B细胞培养和RT-PCR技术从我国人类免疫缺陷病毒1型(HIV-1)感染者中筛选出具有广谱中和活性的人单克隆抗体。方法本研究采集HIV-1感染者抗凝全血,分离出外周血单个核细胞,用磁珠分选纯化记忆性B细胞,在体外选择3T3-hCD40L作为饲养细胞,并使用细胞因子白细胞介素(IL)-2和IL-21诱导记忆性B细胞分泌IgG,用HIV假病毒微量中和的方法进行筛选。从有中和活性的孔中分别扩增出B细胞IgG的轻链和重链基因,并将其克隆到含IgG恒定区基因的载体上。然后将携带重链和轻链基因的质粒进行大量表达,得到人单克隆抗体,并进行抗体结合活性、中和活性及与抗原Gp120亲和力的鉴定。结果从我国3例HIV-1感染者记忆性B细胞中筛选出3株对HIV-1Env抗原有结合活性的单克隆抗体。其中8E7和55B3主要与Gp120结合,19GF3与Gp120和FLSC均可结合且结合活性无明显差异;基因变异大的8E7抗体对A、B、C三种亚型的假病毒均有一定的中和活性,55B3对SF162有中和活性,19GF3未检测到中和活性;这三种单抗的亲和力都很强,8E7、19GF3、55B3与抗原Gp120的KD值分别为6.45×10^(-10) mol/L、1.12×10^(-10) mol/L、2.76×10^(-10) mol/L。结论成功建立了在96孔板中高效刺激人B细胞在体外分泌IgG的方法,并利用HIV假病毒微量中和实验和RT-PCR技术筛选得到对HIV-1具有中和活性的人单克隆抗体。

Objective Combined with B cell sorting and RT-PCR methods, this study is aimed to screen broadly neutralizing antibodies against HIV-1 from virus infected individuals. Methods We adapted a technique for isolating memory B cells from peripheral blood of HIV-1 infected individuals. B cells were cultured with irradiated 3T3-hCD40L feeder cells in medium containing cytokines IL-2 and IL-21. The memory B cells expressing the Env-specific antibody were screened by micro-neutralizing assay. By RT-PCR, Human VH and Vκ or Vλ genes were cloned into eukaryotic expression vectors and then were transfected into 293T cells. The binding and neutralizing activities of antibodies to Env were tested by ELISA and by micro-neutralization assay. In addition, we tested the affinity between the antibody and Gp120. Results Three Env-specific monoclonal antibodies were identified from 3 HIV-1 infected individuals. Two of them could bind with Gp120, the other one bind with both Gp120 and FLSC with similar binding activity. 8E7 had neutralizing activity against three subtypes （A, B, C） of the virus. 55B3 had neutralizing activity only against SF162.19GF3 had no neutralizing activity. The affinities of these three monoclonal antibodies were very strong. The KD values of 8E7, 19GF3, 55B3 to Gp120 protein affinity reached 6.45 × 10^-10 mol/L, 1.12 × 10^（-10） mol/L, 2.76 × 10^（-10） mol/L, respectively. Conclusions We successfully induce B cells to secrete IgG in vitro in 96 well plates. With the HIV pseudovirion neutralization test and RT-PCR technology, neutralizing monoelonal antibodies can be screened.