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10 novembre 2012 6 10 /11 /novembre /2012 07:38
Proteomics-directed cloning of circulating antiviral human monoclonal antibodies Shuji Sato, Sean A Beausoleil, Lana Popova, Jason G Beaudet, Ravi K Ramenani, Xiaowu Zhang, James S Wieler, Sandra M Schieferl, Wan Cheung Cheung & Roberto D Polakiewicz AffiliationsCorresponding authors Nature Biotechnology 30, 1039–1043 (2012) doi:10.1038/nbt.2406 Published online 08 November 2012 In the May 2012 issue of your journal, we described an approach that uses proteomics and next-generation sequencing to identify antigen-specific antibodies directly from the serum of immunized animals, and we applied it to clone circulating antibodies to five different antigens from the serum of rabbits and mice1. Many technologies for isolating antibodies have been developed and applied to gain insight into the specific human antibody response to various pathogens, but none have directly addressed the serological response at the proteomic level2, 3, 4, 5. In addition, recent evidence from a study conducted in mice suggests that not all memory B cells contribute directly to the serological response to a pathogen6. Thus, a method capable of interrogating the composition and complexity of the circulating antibody repertoire elicited to specific vaccines or pathogens is necessary. Here, we report the application of our strategy1 to the identification and cloning of high-affinity, antigen-specific human monoclonal antibodies directly from plasma of a donor vaccinated against hepatitis B virus (HBV). We also clone potent neutralizing human monoclonal antibodies against human cytomegalovirus (HCMV) from a healthy, naturally infected individual. A key first step of our approach is the affinity purification of polyclonal material from a serum or plasma sample to enrich for monoclonal antibodies with desired functional properties. Earlier we have enriched for antibodies with high specific activity in various immunoassays such as enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence, flow cytometry and immunohistochemistry1. But the affinity purification can be tailored to select specifically for high-affinity interactions, potent neutralization or activity in other types of assays. Once the desired properties have been enriched, liquid chromatography tandem mass spectrometry (LC-MS/MS) is used to identify the monoclonal components of the purified fraction by matching to a reference database of antibody variable regions (V regions) produced by next-generation sequencing of the B cell immunoglobulin repertoire of the immunized animal. In contrast to work in hyperimmunized laboratory animals, translating our approach to the isolation of circulating human antibodies faces several potential challenges. For instance, in our earlier work1 we isolated B cells from the spleen of sacrificed animals, but in humans, accessing immunological organs that are highly enriched in B cells, such as the spleen, lymph nodes or even bone marrow, may not be feasible. Furthermore, in most cases humans are not hyperimmunized or maintained in a controlled environment, as would be the case for laboratory animals. In some cases, naturally exposed or convalescent donors might be the preferred or only source of polyclonal serum and B cell–derived genetic material, and therefore high-titer or adequate quantity of material could be limiting. In such cases, the immunogen is not known and thus the appropriate antigen for isolating the desired antibodies needs to be characterized before screening. As a proof of principle of our methodology (Supplementary Fig. 1), we first sought to isolate immunogen-specific antibodies from HBV-vaccinated donors. Chronic HBV infection affects >350 million people, a considerable percentage of whom succumb to hepatic failure or have a high risk of developing hepatocellular carcinoma7. HBV small surface antigen (HBsAg) is the main clinical marker indicating acute or chronic HBV infection, and the recombinant version of this protein has been widely used in vaccine formulations since 1986 (ref. 8). To investigate the serological immune response in HBV vaccine recipients, we screened plasma samples from volunteers who were recently immunized during an in-house HBV vaccination program for reactivity to recombinant HBsAg by ELISA. Of the six volunteers who had received their second HBV immunization dose 7 d before blood collection, donor C037 showed the strongest plasma γ immunoglobulin (IgG) reactivity to the antigen (step A in Supplementary Fig. 1, Supplementary Fig. 2). The vaccine-specific response of this donor was confirmed by ELISA using total IgG purified with Sepharose-conjugated protein G, in which the half-maximal effective concentration (EC50) was <10 μg ml−1. Full text : http://www.nature.com/nbt/journal/v30/n11/full/nbt.2406.html Others: http://www.nature.com/nbt/focus/sequencing2012/index.html?WT.ec_id=NBT-201210

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