Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses.

Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 103-105 sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) percentage of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in (1) properly assessing the statistical significance of repertoire differences, (2) identifying how two repertoires differ, and (3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox' robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether but also where the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center (GC) B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated GC B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically significant differences between non-immunized, vaccine only, and vaccine-plus-adjuvant groups in terms of Ig properties, including IGHV-family usage, SHM percentage, and characteristics of the BCR complementarity-determining region. Most notably, our analyses identified a robust eVLP-specific feature-enhanced IGHV8-family usage in B-cell repertoires. These findings demonstrate the utility of our technique in identifying statistically significant BCR repertoire differences following vaccination. More generally, our approach is potentially applicable to a wide range of studies in infection, vaccination, auto-immunity, and cancer.

Use of AAScatterPlot tool for monitoring the evolution of the hemagglutinin cleavage site in H9 avian influenza viruses.

MOTIVATION: Viruses rapidly evolve due to their error-prone genome replication, and identifying which mutations are selected for during evolution is critical for virus surveillance efforts. Here we introduce a scatter plot tool (AAScatterPlot) that easily shows the selection and avoidance of certain protein mutations based on biochemical properties. We demonstrate its utility for monitoring the evolution of H9 avian influenza viruses from China between 2005 and 2015, particularly at the hemagglutinin (HA) proteolytic cleavage site (PCS) that can affect virus activation and pathogenicity. RESULTS: Given genome sequences, the AAScatterPlot tool compacts into a single plot, information about the hydropathy index, Van der Waals volume, chemical property and occurrence frequency of amino acid residues. The tool also shows the range of residues that could arise from a single point mutation in the genome, which can then be compared against the observed residues to identify mutation constraints. Through this approach, we found that the 2nd position towards the N-terminus side of the HA PCS (P2 position) avoided hydrophobic residues, whereas the P3 position avoided hydrophilic residues. AVAILABILITY AND IMPLEMENTATION: AAScatterPlot is available at https://github.com/WhittakerLab/AAScatterPlot. CONTACT: gary.whittaker@cornell.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages.

Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE: Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.

Image Restoration and Analysis of Influenza Virions Binding to Membrane Receptors Reveal Adhesion-Strengthening Kinetics.

With the development of single-particle tracking (SPT) microscopy and host membrane mimics called supported lipid bilayers (SLBs), stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data obtained by assays such as surface plasmon resonance.

Single-Particle Tracking Shows that a Point Mutation in the Carnivore Parvovirus Capsid Switches Binding between Host-Specific Transferrin Receptors.

Determining how viruses infect new hosts via receptor-binding mechanisms is important for understanding virus emergence. We studied the binding kinetics of canine parvovirus (CPV) variants isolated from raccoons-a newly recognized CPV host-to different carnivore transferrin receptors (TfRs) using single-particle tracking. Our data suggest that CPV may utilize adhesion-strengthening mechanisms during TfR binding and that a single mutation in the viral capsid at VP2 position 300 can profoundly alter receptor binding and infectivity.

BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires.

The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate BRILIA's utility in B-cell repertoire studies related to VDJ gene usage, mechanisms for adenosine mutations, and SHM hot spot motifs. Furthermore, we show that the complete gene usage annotation and SHM identification across the entire CDR3 are essential for studying the B-cell affinity maturation process through immunosequencing methods.

Stochastic fusion simulations and experiments suggest passive and active roles of hemagglutinin during membrane fusion.

Influenza enters the host cell cytoplasm by fusing the viral and host membrane together. Fusion is mediated by hemagglutinin (HA) trimers that undergo conformational change when acidified in the endosome. It is currently debated how many HA trimers, w, and how many conformationally changed HA trimers, q, are minimally required for fusion. Conclusions vary because there are three common approaches for determining w and q from fusion data. One approach correlates the fusion rate with the fraction of fusogenic HA trimers and leads to the conclusion that one HA trimer is required for fusion. A second approach correlates the fusion rate with the total concentration of fusogenic HA trimers and indicates that more than one HA trimer is required. A third approach applies statistical models to fusion rate data obtained at a single HA density to establish w or q and suggests that more than one HA trimer is required. In this work, all three approaches are investigated through stochastic fusion simulations and experiments to elucidate the roles of HA and its ability to bend the target membrane during fusion. We find that the apparent discrepancies among the results from the various approaches may be resolved if nonfusogenic HA participates in fusion through interactions with a fusogenic HA. Our results, based on H3 and H1 serotypes, suggest that three adjacent HA trimers and one conformationally changed HA trimer are minimally required to induce membrane fusion (w = 3 and q = 1).

Influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics.

We report a method for studying membrane fusion, focusing on influenza virus fusion to lipid bilayers, which provides high temporal resolution through the rapid and coordinated initiation of individual virus fusion events. Each fusion event proceeds through a series of steps, much like multistep chemical reaction. Fusion is initiated by a rapid decrease in pH that accompanies the "uncaging" of an effector molecule from o-nitrobenzaldehyde, a photoisomerizable compound that releases a proton to the surrounding solution within microseconds of long-wave ultraviolet irradiation. In order to quantify pH values upon UV irradiation and uncaging, we introduce a simple silica nanoparticle pH sensor, useful for reporting the pH in homogeneous nanoliter volumes under conditions where traditional organic dye-type pH probes fail. Subsequent single-virion fusion events are monitored using total internal reflection fluorescence microscopy. Statistical analysis of these stochastic events uncovers kinetic information about the fusion reaction. This approach reveals that the kinetic parameters obtained from the data are sensitive to the rate at which protons are delivered to the bound viruses. Higher resolution measurements can enhance fundamental fusion studies and aid antiviral antifusogenic drug development.