Screening Of Fully Human Anti-anthrax Lethal Factor Neutralizing Antibody From Transgenic Mice

Anthrax, which has a wide distribution in the world, is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The Communicable Disease Prevention Act lists anthrax as one of the class B infectious diseases. According to the route of infection, anthrax can be divided into three types, Which are anthrax, gastrointestinal anthrax and pulmonary anthrax. The lethal rate of pulmonary anthrax is as high as 90%, so China treat it as the class A infectious diseases. The causative agent of Bacillus anthracis is consists of three toxin and one poly-D-glutamyl capsule. The three toxin, including protective antigen(PA), lethal factor(LF) and edema factor(EF), are the major pathogenic factor. These proteins alone do not have the toxicity. But if PA and LF combine to form a lethal toxin LT, or PA and EF combine to form an edema toxin ET, after entering cells by receptor-mediated endocytosis, they would cause certain damages to the host cells. Since injuries and deaths of the infected are mainly caused by lethal toxin(LT), antibiotics are hard to work in advanced anthrax infections. Thus, therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies(MAbs) for PA. And US FDA has approved ABTHRAX humanized PA monoclonal antibody to be used for the treatment of inhalational anthrax. Once B. anthracis were artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore anti-LF MAbs is an important supplement for the anthrax treatment.Currently most of the current anthrax anti-LF MAbs are murine MAbs or chimeric MAbs. However the clinical application of murine monoclonal antibodies is limited, due to its short half-life, high immunogenicity and easily causing human anti mouse antibody(HAMA) response. Fully human MAbs have been widely used to avoid the high immunogenicity of murine antibodies, with advantages of long half-life and high safety. With a single B cell antibody preparation techniques emerging, single-cell PCR technique is used for in vitro cloning and expression of a single B cell antibody genes, while the single-cell PCR technology avoids multiple rounds of screening process, obtains antibody gene fast and keeps the natural pairing of the antibody heavy chain and light chain, single-cell PCR technology will become one of the newest methods to obtain fully human monoclonal antibody. The LF is not a component of approved vaccine and it can not be directly immunized to volunteers to obtain fully human MAbs, but transgenic mouse technology can overcome the shortcomings such as no vaccine or no patient blood sample, and transgenic mice can be immunized with specific antigen directly. They can be used to screen fully human monoclonal antibodies. Therefore we can use transgenic mouse technology, single cell sorting technology and single-cell PCR technology to screen fully human anti-LF MAbs.By preparing and purifying LF antigen, and using mouse macrophages J774 A.1 to evaluate the biological activity of LF, LF with high purity, good bio-activity was obtained, which provided guarantees for transgenic mice immune and neutralizing antibody assay.Since there are differences between the immune response of transgenic mice and normal mice, both transgenic mice and normal male C57BL/6 mice were immunized at week 0, 2, 4. The titers of antibody against LF in serum were detected by ELISA. Results showed that the immune responses of transgenic mice and ordinary mice were different. After immuniziation, the antibody titer of transgenic mice increased more slowly than ordinary mice. And the antibody titer of transgenic mice was lower than ordinary mice. Freund’s adjuvant and the antigen mixed together and two transgenic mice were immunized at week 0, 2, 4. Blood were drawed at day 0、7、14、28、42. The LF neutralizing antibody serum titers were detected at different time point by ELISA and TNA.Since there are BCR receptors on the memory B cell surface, LF monoclonal antibodies can be screened by labeling LF-specific memory B cells. 14 days after the last immunization, the spleen cell of transgenic mice were prepared, 288 individual memory B cells were obtained by flow cytometry sorter sorting. Using single cell RTPCR and nested-PCR, 48 pairs of the variable region genes of antibodies were successfully obtained.By overlap extension PCR, 48 pairs of linear antibody gene expression cassettes were constructed and transfected into HEK 293 T cells. Then the supernant were detected by ELISA and 2 binding m Abs were found, respectively named 1D7 and 2B9. The gene sequences were analysed, both 1D7 and 2B9 are are from human germline genes. The expression plasmids of LF m Abs were constructed and transiently transfected into Expi 293 F cells. The affinity of 1D7 and 2b9 were Detected by SPR, m Ab 1D7 had higher affinity than m Ab 2B9. Toxin neutralizion assay showed that these two LF m Abs protected cells very well in vitro, and both m Abs prolonged the survival time of rats in vivo.At last, the combination effects of LF m Abs and PA m Abs were analysed. Results showed that both two LF m Abs could play a synergistic protective effect with PA m Abs. When two LF m Abs were used in combination with no protective PA m Abs 8A7, they produced good protective effects both in vitro and in vivo.In summary, this study successfully obtained anti-LF fully human antibodies by transgenic mouse immunization with LF antigen, and explored the immunization characteristics of transgenic mice and the combination effects of different m Abs. The innovation of this study lies in combining the advantages of transgenic mice, flow sorting and single cell PCR technology. This study not only obtained a fully human anthrax LF monoclonal antibody, but also opened up new ideas and methods for the rapid screening of other fully human monoclonal antibodies.