Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations.

Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.

Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality.

BACKGROUND: We have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features. RESULTS: Here we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue. CONCLUSIONS: In summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.

Salmonella Typhi Vi capsule prime-boost vaccination induces convergent and functional antibody responses.

Vaccine development to prevent Salmonella Typhi infections has accelerated over the past decade, resulting in licensure of new vaccines, which use the Vi polysaccharide (Vi PS) of the bacterium conjugated to an unrelated carrier protein as the active component. Antibodies elicited by these vaccines are important for mediating protection against typhoid fever. However, the characteristics of protective and functional Vi antibodies are unknown. In this study, we investigated the human antibody repertoire, avidity maturation, epitope specificity, and function after immunization with a single dose of Vi-tetanus toxoid conjugate vaccine (Vi-TT) and after a booster with plain Vi PS (Vi-PS). The Vi-TT prime induced an IgG1-dominant response, whereas the Vi-TT prime followed by the Vi-PS boost induced IgG1 and IgG2 antibody production. B cells from recipients who received both prime and boost showed evidence of convergence, with shared V gene usage and CDR3 characteristics. The detected Vi antibodies showed heterogeneous avidity ranging from 10 µM to 500 pM, with no evidence of affinity maturation after the boost. Vi-specific antibodies mediated Fc effector functions, which correlated with antibody dissociation kinetics but not with association kinetics. We identified antibodies induced by prime and boost vaccines that recognized subdominant epitopes, indicated by binding to the de­O-acetylated Vi backbone. These antibodies also mediated Fc-dependent functions, such as complement deposition and monocyte phagocytosis. Defining strategies on how to broaden epitope targeting for S. Typhi Vi and enriching for antibody Fc functions that protect against typhoid fever will advance the design of high-efficacy Vi vaccines for protection across diverse populations.

Molecular definition of multiple sites of antibody inhibition of malaria transmission-blocking vaccine antigen Pfs25.

The Plasmodium falciparum Pfs25 protein (Pfs25) is a leading malaria transmission-blocking vaccine antigen. Pfs25 vaccination is intended to elicit antibodies that inhibit parasite development when ingested by Anopheles mosquitoes during blood meals. The Pfs25 three-dimensional structure has remained elusive, hampering a molecular understanding of its function and limiting immunogen design. We report six crystal structures of Pfs25 in complex with antibodies elicited by immunization via Pfs25 virus-like particles in human immunoglobulin loci transgenic mice. Our structural findings reveal the fine specificities associated with two distinct immunogenic sites on Pfs25. Importantly, one of these sites broadly overlaps with the epitope of the well-known 4B7 mouse antibody, which can be targeted simultaneously by antibodies that target a non-overlapping site to additively increase parasite inhibition. Our molecular characterization of inhibitory antibodies informs on the natural disposition of Pfs25 on the surface of ookinetes and provides the structural blueprints to design next-generation immunogens.

Development and clinical validation of an in situ biopsy-based multimarker assay for risk stratification in prostate cancer.

PURPOSE: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. EXPERIMENTAL DESIGN: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish "favorable" versus "nonfavorable" pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). RESULTS: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on "false-negative" and "false-positive" rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non-GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95). CONCLUSIONS: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy.

Error tolerant indexing and alignment of short reads with covering template families.

The rapid adoption of high-throughput next generation sequence data in biological research is presenting a major challenge for sequence alignment tools­specifically, the efficient alignment of vast amounts of short reads to large references in the presence of differences arising from sequencing errors and biological sequence variations. To address this challenge, we developed a short read aligner for high-throughput sequencer data that is tolerant of errors or mutations of all types­namely, substitutions, deletions, and insertions. The aligner utilizes a multi-stage approach in which template-based indexing is used to identify candidate regions for alignment with dynamic programming. A template is a pair of gapped seeds, with one used with the read and one used with the reference. In this article, we focus on the development of template families that yield error-tolerant indexing up to a given error-budget. A general algorithm for finding those families is presented, and a recursive construction that creates families with higher error tolerance from ones with a lower error tolerance is developed.

Quantification of the yeast transcriptome by single-molecule sequencing.

We present single-molecule sequencing digital gene expression (smsDGE), a high-throughput, amplification-free method for accurate quantification of the full range of cellular polyadenylated RNA transcripts using a Helicos Genetic Analysis system. smsDGE involves a reverse-transcription and polyA-tailing sample preparation procedure followed by sequencing that generates a single read per transcript. We applied smsDGE to the transcriptome of Saccharomyces cerevisiae strain DBY746, using 6 of the available 50 channels in a single sequencing run, yielding on average 12 million aligned reads per channel. Using spiked-in RNA, accurate quantitative measurements were obtained over four orders of magnitude. High correlation was demonstrated across independent flow-cell channels, instrument runs and sample preparations. Transcript counting in smsDGE is highly efficient due to the representation of each transcript molecule by a single read. This efficiency, coupled with the high throughput enabled by the single-molecule sequencing platform, provides an alternative method for expression profiling.

Single-molecule DNA sequencing of a viral genome.

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

SST: an algorithm for finding near-exact sequence matches in time proportional to the logarithm of the database size.

MOTIVATION: Searches for near exact sequence matches are performed frequently in large-scale sequencing projects and in comparative genomics. The time and cost of performing these large-scale sequence-similarity searches is prohibitive using even the fastest of the extant algorithms. Faster algorithms are desired. RESULTS: We have developed an algorithm, called SST (Sequence Search Tree), that searches a database of DNA sequences for near-exact matches, in time proportional to the logarithm of the database size n. In SST, we partition each sequence into fragments of fixed length called 'windows' using multiple offsets. Each window is mapped into a vector of dimension 4(k) which contains the frequency of occurrence of its component k-tuples, with k a parameter typically in the range 4-6. Then we create a tree-structured index of the windows in vector space, with tree-structured vector quantization (TSVQ). We identify the nearest neighbors of a query sequence by partitioning the query into windows and searching the tree-structured index for nearest-neighbor windows in the database. When the tree is balanced this yields an O(logn) complexity for the search. This complexity was observed in our computations. SST is most effective for applications in which the target sequences show a high degree of similarity to the query sequence, such as assembling shotgun sequences or matching ESTs to genomic sequence. The algorithm is also an effective filtration method. Specifically, it can be used as a preprocessing step for other search methods to reduce the complexity of searching one large database against another. For the problem of identifying overlapping fragments in the assembly of 120 000 fragments from a 1.5 megabase genomic sequence, SST is 15 times faster than BLAST when we consider both building and searching the tree. For searching alone (i.e. after building the tree index), SST 27 times faster than BLAST. AVAILABILITY: Request from the authors.