Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases.

Weixu Meng,Ling Chen,Liqiang Feng,Caijun Sun,Weiqi Pan,Guowei Wei,Zhengfeng Li,Zhenyuan Dong,Kwok-Yung Yuen,Qinfang Luo,Xiangjing Hu,Nanshan Zhong,Anna J X Zhang,Chufang Li,Shibo Jiang

doi:10.1371/journal.pone.0066276

Abstract

BackgroundThe outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs).Methodology/Principal FindingsThe genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.Conclusions/SignificanceOur study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.

Highlights

Outbreaks of infectious diseases, such as the severe acute respiratory syndrome (SARS) epidemic in 2003 and several influenza pandemics especially H5N1, H1N1, and most recently the emergent cases of H7N9, have caused loss of human life, public panic, and economic setbacks
We demonstrated an integrated process as follows: 1) the use of recombinant adenoviral vectors carrying synthetic genes encoding the target antigens allowed the rapid preparation of antigens without the need for having the pathogen in hand; 2) the immunization of rhesus macaques (Macaca Mulata) with the vectored antigen or the pathogen, if available; 3) the screening of an antibody phage display library derived from B cells harvested from immunized macaques; 4) cloning the macaque immunoglobulin heavy chain and light chain variable regions and combining them with human constant regions to generate human-macaque chimeric monoclonal antibodies (mAbs); and 5) further characterization of the mAbs using in vitro and in vivo studies
The human-like mAbs are a good alternative in the event of a virulent infectious outbreak, especially if there are no human survivors from the infection or ethical and regulatory issues arise that prevent the immunization of individuals with an antigen or a pathogen

Summary

Introduction

Outbreaks of infectious diseases, such as the severe acute respiratory syndrome (SARS) epidemic in 2003 and several influenza pandemics especially H5N1, H1N1, and most recently the emergent cases of H7N9, have caused loss of human life, public panic, and economic setbacks. Vaccines against specific pathogens are the most effective means of protecting humans from infection. It has been shown that individuals who recover from H5N1 or H1N1 viral infections can generate neutralizing antibodies against the pathogen, and their plasma confers therapeutic protection in infected individuals when administered passively [1,2]. A method to rapidly generate and select neutralizing antibodies is urgently needed for the defense against new virulent pathogens. The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Conclusions/Significance: Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases

Methods

Results

Conclusion