Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris

Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, ‘hit-to-lead identification’ remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigenspecific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).Bill & Melinda Gates FoundationUnited States. Defense Advanced Research Projects AgencySpace and Naval Warfare Systems Center San Diego (U.S.) (Contract N66001-13-C-4025)W. M. Keck FoundationNational Institute of Allergy and Infectious Diseases (U.S.) (U19AI090970).National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) Grant P30-CA14051

Profiling Human Antibody Responses by Integrated Single-Cell Analysis

Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform, using arrays of subnanoliter wells (nanowells), for constructing detailed profiles for human B cells comprising the immunophenotypes of these cells, the distribution of isotypes of the secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colon biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments

Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity

Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

Background The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Results Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. Conclusion We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.Peer reviewe

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

BackgroundThe Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function.ResultsHere, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory.ConclusionWe conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.</p

Profiling human antibody responses by integrated single-cell analysis

Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform, using arrays of subnanoliter wells (nanowells), for constructing detailed profiles for human B cells comprising the immunophenotypes of these cells, the distribution of isotypes of the secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colon biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments.W. M. Keck FoundationInternational AIDS Vaccine Initiative (Innovation Award)National Institutes of Health (U.S.)/National Institute of Allergy and Infectious Diseases (U.S.) (U19AI090970)United Negro College Fund-Merck Graduate Research Dissertation Fellowshi

Characterization of relationships between intracellular and secreted proteins for single cells of <i>P. pastoris</i>.

<p>Density plots of the relative rates of eGFP secretion by single cells analyzed by microengraving with respect to the relative amount of intracellular eGFP determined by fluorescence microscopy for clones containing (A) one eGFP gene copy under pGAPDH or (B) two eGFP gene copies under pAOX1. Dashed line indicates the limit of detection for secreted eGFP in microengraving (background+2σ). The median amounts of internal eGFP for cells above and below this limit of detection are marked (X) and are significantly different (Mann-Whitney test, p≪0.001 for both pGAPDH and pAOX strains). (C) Density plot of the relative rates of secretion analyzed by microengraving for the glycosylated Fc fragment and eGFP produced simultaneously in single cells at two different loci using pGAPDH. Pearson's correlation coefficient for secretion of these two proteins as shown is 0.79.</p

Effects of altering relative rates of secretion and degradation on modeled distribution of intracellular and secreted protein.

<p>Density plot of the relative rates of protein secretion by single cells against the relative amount of intracellular protein for model data sets under three conditions: 1) where median k_ERAD≥k_sec (purple), 2) where median k_sec≪k_sec in Condition 1 (light pink), and 3) where median k_sec≪k_sec in Condition 1 and median k_ERAD>k_ERAD and/or k_expexp in Condition 1 (dark pink). The median amount of intracellular protein for populations in Conditions 1 and 3 are marked (X). The secretion-inhibited population (light pink) was generated by increasing t_sec to 400 min (standard deviation 40 min) while keeping all other parameters the same as the initial population (purple) derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037915#pone-0037915-g004" target="_blank">Figure 4C</a>. The stress-induced population (dark pink) was generated by reducing k_exp or decreasing t_ERAD to obtain a similar median level of protein in the ER as the original population.</p

Analysis of steady-state distributions of rates of secretion.

<p>(A) Distributions of rates of secretion of eGFP for (Top) a clone with a single copy of eGFP under transcriptional control of pGAPDH, and (Bottom) a clone with two copies of eGFP under transcriptional control of pAOX1. Red squares indicate binned single-cell secretion events following microengraving with each clone. Blue lines show the best fits using Eq. (1). Values for <i>a</i> and <i>b</i> are shown. (B) Relationship between <i>a</i> (burst frequency) and <i>b</i> (burst size) for proteins expressed using either pGAPDH (top) or pAOX1 (bottom) as a function of gene copy number and complexity. Clones secreting eGFP (green), clones secreting aglycosylated Fc fragment (blue) and clones secreting glycosylated Fc fragment (red) are shown for a single gene copy (squares), 2–3 gene copies (triangles) and 4 or more gene copies (circles). Error bars represent S.E.M. for each clone from at least three separate measurements.</p

<i>Pichia pastoris</i> strains generated for systematic investigation of the relationship between protein complexity, gene dosage, relative expression and secretion.

a<p>Strain names indicate promoter used.</p>b<p>Determined by qPCR using an absolute quantification of transcript copy number.</p>c<p>Determined by RT-qPCR using an absolute quantification of transcript copy number and the expression of actin as a control.</p