Ebola: surprisingly Little immune data


http://www.nbcnews.com – A Transmission Electron Micrograph (TEM) of the Ebola virus RNA

Ebola is a clearly under-studied disease as no vaccine or treatment exists. We, at IEDB, in turn, were also surprised to see how little data on Ebola epitopes were reported to date. It took us just a couple of days to report on available data and a bit longer to get an attention (well, I am not good at publicity as I write this in a month since we published the first report, while Ebola is not getting away, and we are working on a larger publication).

To summarize, the EBOV genome encodes seven proteins: envelope glycoprotein (GP), nucleoprotein (NP), polymerase cofactor (VP35), transcription activator (VP30), matrix protein (VP40), secondary matrix protein (VP24), and RNA polymerase (L). There were reported 44 T cell and 15 B cell epitopes for GP (the surface protein and the most important target for antibodies) and 10 T cell and 20 B cell epitopes for NP, with a far fewer epitopes reported for other proteins. Unexpectedly, human and non-human primate hosts were absent in the T and B-cell assays, with murine hosts predominated and a smaller number of rabbit studies reported only for antibodies.

What was also surprising to us that of the monoclonal antibodies (mAbs) described to date, only one was derived from a human survivor of Ebola disease of the 1995 Kikwit outbreak (mAb KZ52). Only nine mAbs showed to be protective in mice (just recently, a new study using macaque was published in Nature) or in in vitro virus neutralization assays. Six of these mAbs comprise the three cocktails, ZMab, ZMapp, and MB-003, that have being evaluated as treatment options for Ebola disease, and ZMapp was presumably contributed to the successful treatment of two patients in the US. There were reported efforts to increase the Zmapp production after the already mentioned publication in Nature (as FDA clears an Ebola therapeutic approval for human if it was successfully tested in macaque).

It should be expected that the current attention to Ebola will result in discovering new protective human antibodies and immunodominant epitopes from survivors of the current outbreak, which can be used as therapeutics.

[When I was about to click submit, my husband asked me what this my post had to do with BigBioData. So I capitalized “Little” in the title — sometimes we wish to have Big Data, but we don’t realize that acquiring them might cost lives as it was with collecting and sequencing Ebola strains in current outbreak.]



Is it time for sequencing entire antibody repertoires?


Continuing on the theme of immunosequencing, this post is about Atreca, Inc., founded in 2010 in San Carlos, California, and funded by the Bill & Melinda Gates Foundation. I first heard about this company a year ago from its co-founder Prof. Robinson of Stanford. As he explained in his talk given at Scripps, the company utilizes a novel HT technology, called Immune Repertoire Capture™, allowing to isolate B cells (plasmablasts, or plasma B cells producing antibodies, precisely), to barcode and sequence their cDNA, and finally to perform bioinformatics analysis (building a tree from comparison of antibody chain sequences). In the result, in two weeks, the whole antibody repertoire of an individual is decoded, and most importantly the pairing between light and heavy chains of each antibody is established because the technology allows to barcode each cell individually. That allows to get the whole makeup of an in antibody repertoire, frozen in time, and to see the rare clone families of antibodies, immunodominant (recognizing specific antigens) antibodies and also memory B cells, which would look like single branches on the dendogram of all antibodies (or B-cells, which is the same here as each B cell produces one type of antibody).

Applied to human disease, Immune Repertoire Capture™ is an engine for the discovery and development of antibody-based therapeutics, vaccines, diagnostics, and research reagents in therapy areas including cancer, infectious disease, and autoimmune disease.

How could it be? One example, take a cancer patient who is a long term non-progressor and look into which unique antibodies she produces that progressors do not — those antibodies can be studied to become antibody therapeutics against that form of cancer.

How is the company doing today? Unfortunately, not much info can be found in the news for the last year, except about 3 rounds of financing, two of which are debt financings, and a job ad on BioSpace for a Research Associate, versed in PCR and NGS. The company website is dated by 2012. Searching for Atreca, LinkedIn returns 20 profiles. The concept looks right and timely. Or, overwhelmed by genomics data, the world is not ready to deal with yet another deluge of big data?

ImmunoSequencing holds great future for immunotherapy


Adaptive Biotechnologies is doing something unimaginable 4-5 years ago by adding on top of genomics, proteomics, microbiomics and metabolomics data yet another layer of big biological data.

“Adaptive” refers to the adaptive immune system, the major players of which are B and T cells as they are the major types of lymphocytes. The human body has about 2 trillion lymphocytes, constituting 20–40% of white blood cells and weighting about the same as the brain or liver (!). The peripheral blood contains 2% of circulating lymphocytes; the rest move within the tissues and lymphatic system. All vertebrate animals (or Chordata) — amphibiansreptiles,mammals, and birds — have an adaptive immune system, while other living organisms do not.

Our body, specifically bone marrow and thymus, generate every day 10^8 – 10^12 T and B cells, respectively, each carrying a unique B and T cell receptor (called BCR and TCR), made by selecting and splicing together V, D and J genes from an individual’s genome. At the junctions between V-D and D-J segments, a varying number of nucleotides are deleted and a special enzyme inserts random nucleotides, creating a unique TCR or BCR. B and T cells circulate throughout the lymphatic system for a month or two recognizing pathogens and becoming in the result memory B and T cells so next time the body encounter the same pathogen it can be killed immediately. Every individual has a unique repertoire, or “makeup”, of B and T cells circulating in her body, which is also unique to the current immune status of the individual. Thus it will differ when the person becomes sick. It is not hard to imagine the medical applications: for example, B cells produce antibodies, and thus the B cell repertoire of patients who fought HIV or cancer present an interest for finding new antibody therapeutics against HIV and cancer.

Adaptive Biotechnologies developed a technology, based on massive parallel sequencing of T cell receptors, that allows to  identify 10-15 million unique TCRs in one individual. “The immunoSEQ assay utilizes a multiplex PCR strategy to amplify the CDR3 region of the T cell receptor, spanning the variable region formed by the junction of the V, D and J segments and their associated non-templated insertions. In most cases, the identity of each segment is also captured. The resulting 60 base pair nucleotide sequence may be used as an identifier or “tag” for a particular clone across different samples.” (from the company website)

Current focus of the company on a drug therapy targeting the specific TCR combinations associated with prevalent auto-immune diseases, when T cells mistakenly attack the proteins of the individual. Thus, in collaboration with the Benaroya Research Institute at Virginia Mason Hospital in Seattle, Adaptive is currently screening blood samples from patients with either Type 1 Diabetes or Multiple Sclerosis for public TCR sequences. Over the next 12 months, Adaptive plans to develop general diagnostics to use as clinical measurements, with an initial focus in oncology. The company already offers ClonoSEQ, a set of CLIA-standardized assays developed for highly sensitive (10^6) detection of Minimal Residual Disease in leukemia and lymphoma patients.

The company provides the full service, so researchers can ship DNA and cDNA samples to Adaptive’s headquarters in Seattle, Washington, and then interpret and manipulate produced data on their computers using a suite of cloud-based software tools called the immunoSEQ Analyzer.