Research Of Fully Human Monoclonal Antibodies Against Hantavirus Based On Single B Cell

Haemorrhagic fever with renal syndrome(HFRS) is an acute infectious disease caused by Hantaviruses of the Hantavirus genus, family Bunyaviridae. It is clinically characterized in humans by fever, haemorrhage, headache, abdominal pain, and acute kidney damage. In severe cases, death can occur as the result of vascular leakage leading to low blood pressure, acute shock and renal failure. More than 100,000 cases of HFRS with a mortality rate of 2-10% occur annually in worldwide. HFRS occurs primarily in the Old World particularly in eastern Asia. China has the highest incidence of HFRS, approximately 90% of global HFRS cases in the last few decades. During recent decades, the incidence of HFRS has fluctuated, but 30,000-60,000 cases are reported annually. There is an increasing number of HFRS cases with more complication, including multiple organ dysfunction syndrome, intracranial hemorrhage and disseminated intravascular coagulation which are the primary causes of death. So it has remained one of the top nine communicable diseases in mainland China.The treatment of HFRS is based on the clinical symptoms of the disease and occasionally includes haemodialysis, oxygenation, and shock therapy. There is no specific therapy available. Early in disease, treatment with ribavirin, an antiviral agent, is associated with a reduction in morbidity and a decrease in fatalities from HFRS in China. Other promising new measures, including the use of a bradykinin receptor antagonist(bradykinin is involved in vasodilatation and increases vascular permeability), mainly based on similar findings in the hamster model, have not been used widely in humans. Steroid-based anti-inflammatory treatment options have been described in several case reports, particularly in patients with prolonged and non-resolving renal failure. However, there has been no clinical trial to confirm the benefit of glucocorticoid treatment. Passive immune therapy with human plasma has been certified in the hamster model and used in humans. There is a worldwide consensus that neutralizing antibody is effective in treatment of HFRS in the fields of clinical treatment and biopharmacy.Development of the PCR methods and maturation of antibody production have made it possible to generate m Abs from single human B cell by single cell RT-PCR with successional cloning and expression in vitro. Compared to traditional monoclonal antibody technologies, single B cell technologies require relatively fewer cells, and are highly efficient in obtaining specific m Abs in a rapid way with preservation of the natural heavy and light chain pairing. With so many advantages, single B cell technologies have been proved to be attractive approaches for retrieval of naive and antigen-experienced antibody repertoires generated in vivo.In order to change the situation that there is no therapeutic antibody of HFRS, this study try to prepare anti-hantavirus fully human neutralizing antibodies based on single B cell of hemorrhagic fever with renal syndrome convalescent patients.First, 80 m L peripheral blood was collected from four HFRS convalescent patients, and lymphocytes were isolated from peripheral blood by density gradient centrifugation within 24 hours of collection. Then the lymphocytes were stained with fluorescence and sorted by flow cytometry. The cells were bulkly batchedly sorted by first gating on CD19high/CD20 low to neg/CD3 neg and then on CD27high/CD38 high cells. Total 2200 B cells were achieved.Next, the antibody genes in each cell were got through two steps. In first step, the VH and VL genes were amplified in one RT-PCR reaction with a cocktail of 18 primers, which were designed due to the diversity of unknown antibody genes that covered all of the families of variable genes possible. In second step, we used the above RT-PCR products as templates to amplify the H chain including VDJ genes, κ chain and λ chain including VJ genes by nested PCR with highly specific primers for each variable gene family. Finally, 130 natural paired antibody genes were obtained from 2200 B cells, in which there were 427 heavy, 880 κ and 458 λ chain variable genes, with positive rate nearly 20%, 40% and 21% respectively. Vκ acounted for 66% while Vλ acounted for 34% in the light chain genes. This percentage basically correlated with the situation of B cells that express κ chain antibody and λ chain antibody.Cloning and expressing of 130 antibodies in mammalian cells are time-consuming, so the 130 Ig G antibodies were expressed by linear gene expression cassette in order to facilitate high throughput test of the Ig VH and VL. Each linear full-length heavy and light chain gene expression cassettes were assembled by overlapping PCR with 3 DNA fragments, including the C fragment made of the CMV promoter and sequence encoding for an Ig leader, either the VH, Vκ or Vλ genes amplified from single B cells, and either the H fragment made of the IgG1 constant region and BGH poly(A) signal sequence, K fragment made of the Igκ constant region and BGH poly(A) signal sequence, or L fragment made of the Igλ constant region and BGH poly(A) signal sequence. The PCR products of the paired heavy and light chain gene expression cassettes were co-transfected into HEK-293 cells and 120 of 130 antibodies were successfully expressed. Compared with the cloning method, the linear gene expression cassette was work-reducing and the Ig G proteins were expressed within 3-4 days with an average level of 1.2μg /m L.Inactivated hantavirus was used as antigen for detecting the specificity of expressed Ig G antibodies by ELISA. Our results showed that 51 of 120 Ig G were hantavirus-specific. Then, 30 m Abs with higher specificity were cloned and expressed. The neutralizing activities of the purified antibodies were detected by FAVN, which detected the virus-infecting cells by the FITC-labeled anti-hantavirus m Ab. Two antibodies(20C8 and 11H11) with dose-depending neutralizing effects were achieved, and 11H11 had obviously stronger activity than 20C8. Only 0.16 μg of 11H11 was enough to neutralize 70% of 200 TCID50 hantavirus, while 0.64 μg of 20C8 was needed to achieve the same effection. The binding EC50 of 20C8 and 11H11 detected by inactivated hantavirus was about 8.3×10-8 mol/L and 1.8×10-6 mol/L, respectively.In order to find the neutralizing antigen molecules of 20C8 and 11H11, the expression of the G1 and G2 proteins, which are the main neutralizing antigen of hantavirus was attempted in both mammalian cells and Pichia pastoris yeast cells. There is a little of G2 expressed in Pichia pastoris, but G1 did not expressed. Both of G1 and G2 could be expressed in HEK-293 cells that existed in cytomembrane without secreting outside the cells. Lots of work should be done to confirm the epitopes of 20C8 and 11H11, and to evaluate the binding capacity and specificity of these two antibodies.In this study, the method of generating fully human m Abs based on peripheral blood B cells of patients was successfully established. 2200 B cells were catched from 80 m L peripheral blood derived from four HFRS convalescent patients, 130 pairs of heavy chain and light chain genes were amplified by RT-PCR and nested PCR. 120 m Abs were expressed to analyze their specificity by establishing linear gene expression cassettes, 51 anti-hantavirus specific m Abs were achieved. 30 m Abs were tested for neutralizing activity, two of which could neutralize the hantavirus 8205. 0.16 μg of 11H11 was needed to neutralize 70% of 200 TCID50 hantavirus, while 0.64 μg for 20C8. The binding EC50 of 20C8 and 11H11 detected by inactivated hantavirus was about 8.3×10-8 mol/L and 1.8×10-6 mol/L, respectively.The method of generating fully human m Abs based on peripheral blood B cells of patients successfully established in this study is effective to prepare the anti-virus neutralizing antibodies. Preventive and therapeutic antibodies could be achieved from peripheral blood derived from infectious patients or volunteers receiving vaccines. In our study, two anti-hantavirus neutralizing antibodies were screened and assessed, which would be useful for passive immunotherapy for HFRS with deep evaluation and optimization.