Study Of Developing Human Fab Antibody Against SARS Virus

The highly infectious severe acute respiratory syndrome (SARS) which had high mortality rate, was caused by a novel coronavirus, SARS-CoV. So far, there were no . effective anti-SARS drugs and vaccines or therapies available. The development of human anti-SARS monoclonal antibodies would provide a useful passive immunity for the treatment of SARS patients.Phage display technique has become a powerful tool for generation of humanantibodies in recent years. It is easy to manipulate and have the advantage of high selection capacity. It is also a good way to obtain rare antibodies and to obtain completely humanized MAbs directly. This study could be divided into four parts: construction of combinatorial Fab library; enrichment of the Fab library and selection of monoclonal Fab specially against S protein; expression of MIA protein in E.Coli andquantitative analysis of M1A activity.1. The construction of combinatorial Fab libraryThe lymphocytes used for construction of anti-SARS-CoV Fab library were derived from the serum of convalescent SARS patients, in which liters of protective IgG to SARS-CoV were higher than 1: 256 and 1: 64 by ELISA and NT respectively. RNA was extracted from the lymphocytes and the human Fab genes were amplified by RT-PCR. Totally 9 light chain genes and 7 heavy chain genes were obtained. The mixed light and heavy chain genes were cloned into plasmid pComb3. Human Fab combinatorial library with a capacity of about 2×106 cfu was constructed.2. Enrichment of the Fab library and selection of monoclonal Fab specially against S proteinIt is crucial to choose proper target protein to screen the Fab combinatorial library when trying to obtain neutralizing antibodies. Recent studies have demonstrated that the spike protein of SARS-CoV could mediale the altachment of virus to susceptible cells and further induce the cell fusion. Spike protein of SARS-CoV acts as an important antigenic determinant to induce host immune response. Anti-SARS neutralizing antibodies were screened from the Fab library by using S protein as target protein. After 4 rounds of "adsorption- elution- amplification", phage antibodies against S protein have been enriched.Screening of human mAbs from Fab library .included two procedures: determination of binding specificity and screening of neutralizing activity. Firstly, reliable double antibody sandwich ELISA was established to detect the binding specificity of human lab. 12 Fab clones special against S protein were screened from 50 lab clones randomly selected from the Fab library. After testing their neutralizing activity, one clone named M1A was selected, which could postpone the occurrence of CPE in Vero-E6 cells after SARS-CoV challenge. DNA sequence analysis confirmed that the light and heavy chain genes of M1A Fab were belong to the family of V 3a and VH3 respectively.3. Expression of M1A protein in E.Coli.After cloning M1A genes into PET32a vector, the inducing conditions for proper expression were briefly determined by detecting the expression levels in different temperatures and different concentrations of E.coli. After expression of M1A Fab in 200ml culture, a yield of 10mg purified Fab was obtained from the supernatant and pellet by affinity chromatography. A single band was displayed on non-reducing PAGE, which indicated that highly purified solubleM1A protein was obtained after expression and purification.4. Quantitative analysis of the binding and neutralizing activity of M1AThe binding activity of Ml A to S protein was tested by double antibody sandwich ELISA and IFA. Neutralizing test demonstrated that 0.25 mg/ml of Ml A could postpone the occurrence of CPE for 2 days when challenged with 100 TCID50 SARS-CoV, and 0.5 mg/ml of Ml A could totally neutralize the activity of SARS-CoV, with no CPE appeared for 7 days. Real-time fluorescence RT-PCR was utilized to test the difference of SARS-CoV copies between 0.5mg/ml MIA treated group and control group. The results showed that virus copies in Ml A treated group was much lower than control group. So the neutralizing activity of Ml A was further confirmed by molecule biology method.Based on above results, the binding site of Ml A was analyzed roughly. The binding activity of mouse mAb 2C5 to S protein could be inhibited partially, not completely, by M1A when measured with competing ELISA. 2C5 is specifically targeted to S1 domain (aa 350-535, the binding site of SARS-CoV S protein to its receptor angiotensin-converting enzyme 2(ACE 2)). This result suggested that the binding site of M1A might be located near ACE2 receptor.In conclusion, we successfully developed one neutralizing antibody against...