Publisher Correction: Actively personalized vaccination trial for newly diagnosed glioblastoma.

The additional author support information was erroneously omitted from the Supplementary Information. This has been corrected online.

Actively personalized vaccination trial for newly diagnosed glioblastoma.

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.

MCRL: using a reference library to compress a metagenome into a non-redundant list of sequences, considering viruses as a case study.

MOTIVATION: Metagenomes offer a glimpse into the total genomic diversity contained within a sample. Currently, however, there is no straightforward way to obtain a non-redundant list of all putative homologs of a set of reference sequences present in a metagenome. RESULTS: To address this problem, we developed a novel clustering approach called 'metagenomic clustering by reference library' (MCRL), where a reference library containing a set of reference genes is clustered with respect to an assembled metagenome. According to our proposed approach, reference genes homologous to similar sets of metagenomic sequences, termed 'signatures', are iteratively clustered in a greedy fashion, retaining at each step the reference genes yielding the lowest E values, and terminating when signatures of remaining reference genes have a minimal overlap. The outcome of this computation is a non-redundant list of reference genes homologous to minimally overlapping sets of contigs, representing potential candidates for gene families present in the metagenome. Unlike metagenomic clustering methods, there is no need for contigs to overlap to be associated with a cluster, enabling MCRL to draw on more information encoded in the metagenome when computing tentative gene families. We demonstrate how MCRL can be used to extract candidate viral gene families from an oral metagenome and an oral virome that otherwise could not be determined using standard approaches. We evaluate the sensitivity, accuracy and robustness of our proposed method for the viral case study and compare it with existing analysis approaches. AVAILABILITY AND IMPLEMENTATION: https://github.com/a-tadmor/MCRL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer.

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of ß2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Mutant MHC class II epitopes drive therapeutic immune responses to cancer.

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4(+) T cells. Vaccination with such CD4(+) immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4(+) T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient's tumour with vaccines produced 'just in time'.

Spatiotemporal manipulation of retinoic acid activity in zebrafish hindbrain development via photo-isomerization.

All-trans retinoic acid (RA) is a key player in many developmental pathways. Most methods used to study its effects in development involve continuous all-trans RA activation by incubation in a solution of all-trans RA or by implanting all-trans RA-soaked beads at desired locations in the embryo. Here we show that the UV-driven photo-isomerization of 13-cis RA to the trans-isomer (and vice versa) can be used to non-invasively and quantitatively control the concentration of all-trans RA in a developing embryo in time and space. This facilitates the global or local perturbation of developmental pathways with a pulse of all-trans RA of known concentration or its inactivation by UV illumination. In zebrafish embryos in which endogenous synthesis of all-trans RA is impaired, incubation for as little as 5 minutes in 1 nM all-trans RA (a pulse) or 5 nM 13-cis RA followed by 1-minute UV illumination is sufficient to rescue the development of the hindbrain if performed no later than bud stage. However, if subsequent to this all-trans RA pulse the embryo is illuminated (no later than bud stage) for 1 minute with UV light (to isomerize, i.e. deactivate, all-trans RA), the rescue of hindbrain development is impaired. This suggests that all-trans RA is sequestered in embryos that have been transiently exposed to it. Using 13-cis RA isomerization with UV light, we further show that local illumination at bud stage of the head region (but not the tail) is sufficient to rescue hindbrain formation in embryos whose all-trans RA synthetic pathway has been impaired.

Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma.

BACKGROUND: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial mouse tumor, the CT26 colon carcinoma cell line. RESULTS: We found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class II is expressed. Several known cancer-testis antigens are expressed, including Atad2, Cep55, and Pbk. The highest expressed gene is a mutated form of the mouse tumor antigen gp70. Of the 1,688 non-synonymous point variations, 154 are both in expressed genes and in peptides predicted to bind MHC and thus potential targets for immunotherapy development. Based on its molecular signature, we predicted that CT26 is refractory to anti-EGFR mAbs and sensitive to MEK and MET inhibitors, as have been previously reported. CONCLUSIONS: CT26 cells share molecular features with aggressive, undifferentiated, refractory human colorectal carcinoma cells. As CT26 is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies.

Multi-Omics Characterization of the 4T1 Murine Mammary Gland Tumor Model.

Background: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. The 4T1 murine mammary cancer cell line is one of the most widely used breast cancer models. Here, we present an integrated map of the genome, transcriptome, and immunome of 4T1. Results: We found Trp53 (Tp53) and Pik3g to be mutated. Other frequently mutated genes in breast cancer, including Brca1 and Brca2, are not mutated. For cancer related genes, Nav3, Cenpf, Muc5Ac, Mpp7, Gas1, MageD2, Dusp1, Ros, Polr2a, Rragd, Ros1, and Hoxa9 are mutated. Markers for cell proliferation like Top2a, Birc5, and Mki67 are highly expressed, so are markers for metastasis like Msln, Ect2, and Plk1, which are known to be overexpressed in triple-negative breast cancer (TNBC). TNBC markers are, compared to a mammary gland control sample, lower (Esr1), comparably low (Erbb2), or not expressed at all (Pgr). We also found testis cancer antigen Pbk as well as colon/gastrointestinal cancer antigens Gpa33 and Epcam to be highly expressed. Major histocompatibility complex (MHC) class I is expressed, while MHC class II is not. We identified 505 single nucleotide variations (SNVs) and 20 insertions and deletions (indels). Neoantigens derived from 22 SNVs and one deletion elicited CD8+ or CD4+ T cell responses in IFNγ-ELISpot assays. Twelve high-confidence fusion genes were observed. We did not observe significant downregulation of mismatch repair (MMR) genes or SNVs/indels impairing their function, providing evidence for 6-thioguanine resistance. Effects of the integration of the murine mammary tumor virus were observed at the genome and transcriptome level. Conclusions: 4T1 cells share substantial molecular features with human TNBC. As 4T1 is a common model for metastatic tumors, our data supports the rational design of mode-of-action studies for pre-clinical evaluation of targeted immunotherapies.

A coarse-grained biophysical model of E. coli and its application to perturbation of the rRNA operon copy number.

We propose a biophysical model of Escherichia coli that predicts growth rate and an effective cellular composition from an effective, coarse-grained representation of its genome. We assume that E. coli is in a state of balanced exponential steady-state growth, growing in a temporally and spatially constant environment, rich in resources. We apply this model to a series of past measurements, where the growth rate and rRNA-to-protein ratio have been measured for seven E. coli strains with an rRNA operon copy number ranging from one to seven (the wild-type copy number). These experiments show that growth rate markedly decreases for strains with fewer than six copies. Using the model, we were able to reproduce these measurements. We show that the model that best fits these data suggests that the volume fraction of macromolecules inside E. coli is not fixed when the rRNA operon copy number is varied. Moreover, the model predicts that increasing the copy number beyond seven results in a cytoplasm densely packed with ribosomes and proteins. Assuming that under such overcrowded conditions prolonged diffusion times tend to weaken binding affinities, the model predicts that growth rate will not increase substantially beyond the wild-type growth rate, as indicated by other experiments. Our model therefore suggests that changing the rRNA operon copy number of wild-type E. coli cells growing in a constant rich environment does not substantially increase their growth rate. Other observations regarding strains with an altered rRNA operon copy number, such as nucleoid compaction and the rRNA operon feedback response, appear to be qualitatively consistent with this model. In addition, we discuss possible design principles suggested by the model and propose further experiments to test its validity.

Mutated tumor alleles are expressed according to their DNA frequency.

The transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. In conclusion, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency.

Probing individual environmental bacteria for viruses by using microfluidic digital PCR.

Viruses may very well be the most abundant biological entities on the planet. Yet neither metagenomic studies nor classical phage isolation techniques have shed much light on the identity of the hosts of most viruses. We used a microfluidic digital polymerase chain reaction (PCR) approach to physically link single bacterial cells harvested from a natural environment with a viral marker gene. When we implemented this technique on the microbial community residing in the termite hindgut, we found genus-wide infection patterns displaying remarkable intragenus selectivity. Viral marker allelic diversity revealed restricted mixing of alleles between hosts, indicating limited lateral gene transfer of these alleles despite host proximity. Our approach does not require culturing hosts or viruses and provides a method for examining virus-bacterium interactions in many environments.