Machine Learning Identifies Stemness Features Associated with Oncogenic Dedifferentiation.

Cancer progression involves the gradual loss of a differentiated phenotype and acquisition of progenitor and stem-cell-like features. Here, we provide novel stemness indices for assessing the degree of oncogenic dedifferentiation. We used an innovative one-class logistic regression (OCLR) machine-learning algorithm to extract transcriptomic and epigenetic feature sets derived from non-transformed pluripotent stem cells and their differentiated progeny. Using OCLR, we were able to identify previously undiscovered biological mechanisms associated with the dedifferentiated oncogenic state. Analyses of the tumor microenvironment revealed unanticipated correlation of cancer stemness with immune checkpoint expression and infiltrating immune cells. We found that the dedifferentiated oncogenic phenotype was generally most prominent in metastatic tumors. Application of our stemness indices to single-cell data revealed patterns of intra-tumor molecular heterogeneity. Finally, the indices allowed for the identification of novel targets and possible targeted therapies aimed at tumor differentiation

Antibody Neutralization of an Influenza Virus that Uses Neuraminidase for Receptor Binding

Influenza virus infection elicits antibodies against the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Because HA is essential for viral entry, antibodies targeting HA often potently neutralize the virus in single-cycle infection assays. However, antibodies against NA are not potently neutralizing in such assays, since NA is dispensable for single-cycle infection. Here we show that a modified influenza virus that depends on NA for receptor binding is much more sensitive than a virus with receptor-binding HA to neutralization by some anti-NA antibodies. Specifically, a virus with a receptor-binding G147R N1 NA and a binding-deficient HA is completely neutralized in single-cycle infections by an antibody that binds near the NA active site. Infection is also substantially inhibited by antibodies that bind NA epitopes distant from the active site. Finally, we demonstrate that this modified virus can be used to efficiently select mutations in NA that escape antibody binding, a task that can be laborious with typical influenza viruses that are not well neutralized by anti-NA antibodies. Thus, viruses dependent on NA for receptor binding allow for sensitive in vitro detection of antibodies binding near the catalytic site of NA and enable the selection of viral escape mutants

Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms

Linkage analysis of high frequency variants that emerged during rSV12-HA passage.

<p><b>(A)</b> Graphical representation of genetic linkage between selected variants within the three parallel passage 3 rSV12-HA populations, based on PrimerID sequencing. Node size is proportional to relative frequency of the indicated allele across the three populations. Aqua nodes indicate parental amino acids and red nodes represent variant amino acids. Edges connect pairs of amino acids that were linked in greater than 0.02% of all primerID consensus reads examined. <b>(B)</b> Multi-step growth comparison of the indicated viruses in eggs.</p

PrimerID sequencing of HA genes from recombinant WT and SV12-HA following passage.

<p>Recombinant PR8 (WT) and rSV12-HA (SV12) viruses were rescued via reverse genetics. <b>(A)</b> Amino acid variant frequencies within WT (black) and SV12 HA (red) populations collected from rescue supernatants without amplification, as determined by primerID sequencing. (<b>B,C</b>) Amino acid variant frequencies within WT <b>(B)</b> and rSV12-HA <b>(C)</b> populations following three passages in eggs. For each population, all variants over 0.1% are colored to allow them to be visually distinguished. <b>(D)</b> Comparison of amino acid variant frequencies within rSV12-HA populations before and after 3 passages in eggs. Passage 3 data represents mean +/- SD of three independent passage lines. All amino acid numbering indicated on graphs <b>A-C</b> is from initiating methionine, not according to H3 system.</p

Bio-layer interferometry comparison of binding properties of WT, SV12 and SV12-N133T.

<p>To measure binding properties of WT PR8, SV12, and the compensatory glycan addition mutant N133T, we bound biotinylated 3’SLN to sensor at different concentrations, and measured the K_on and K_off of intact purified virions in the presence and absence of the NA inhibitor oseltamivir, respectively. We fit two-phase (association then dissociation) nonlinear regression curves based on the average of 2–3 experiments.</p

Characterization of compensatory glycan additions.

<p><b>(A)</b> Western blot comparison of HA glycosylation mutants following endoglycosidase treatment. Treatment 1 = undigested, treatment 2 = Endo H digested, treatment 3 = PNGase digested. <b>(B)</b> Growth comparison of PR8, SV12, and SV12 plus individual compensatory mutations in eggs. Eggs were inoculated with 500 TCID50, and viral loads within allantoic cavities were assessed by TCID50 assay at the indicated time points.</p

Fitness tradeoff associated with the accumulation of antigenic escape substitutions in HA.

<p>Growth of plaque purified biological clones of PR8 WT and SV12 was compared in eggs. Eggs were infected in triplicate with 500 TCID50 of the indicated virus. 48 hours later, viral loads in allantoic cavities were titered by TCID50 assay.</p

Correction: Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs

<p>Correction: Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs</p