Infant antibody and B-cell responses following confirmed pediatric GII.17 norovirus infections functionally distinguish GII.17 genetic clusters

Genogroup II (GII) noroviruses are a major cause of diarrheal disease burden in children in both high- and low-income countries. GII.17 noroviruses are composed of distinct genetic clusters (I, II, IIIa, and IIIb) and have shown potential for replacing historically more prevalent GII.4 strains, but the serological basis for GII.17 antigenic diversity has not been studied in children. Utilizing samples from a birth cohort, we investigated antibody and B-cell responses to GII.17 cluster variants in confirmed GII.17 infections in young children as well as demonstrated that the distinct genetic clusters co-circulate. Polyclonal serum antibodies bound multiple clusters but showed cluster-specific blockade activity in a surrogate virus neutralization assay. Antibodies secreted by immortalized memory B cells (MBCs) from an infant GII.17 case were highly specific to GII.17 and exhibited blockade activity against this genotype. We isolated an MBC-derived GII.17-specific Immunoglobulin A (IgA) monoclonal antibody called NVA.1 that potently and selectively blocked GII.17 cluster IIIb and recognized an epitope targeted in serum from cluster IIIb–infected children. These data indicate that multiple antigenically distinct GII.17 variants co-circulate in young children, suggesting retention of cluster diversity alongside potential for immune escape given the existence of antibody-defined cluster-specific epitopes elicited during infection

Viridot: An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus.

The gold-standard method for quantifying neutralizing antibody responses to many viruses, including dengue virus (DENV), is the plaque reduction neutralization test (PRNT, also called the immunofocus reduction neutralization test). The PRNT conducted on 96-well plates is high-throughput and requires a smaller volume of antiserum than on 6- or 24-well plates, but manual plaque counting is challenging and existing automated plaque counters are expensive or difficult to optimize. We have developed Viridot (Viridot package), a program for R with a user interface in shiny, that counts viral plaques of a variety of phenotypes, estimates neutralizing antibody titers, and performs other calculations of use to virologists. The Viridot plaque counter includes an automatic parameter identification mode (misses <10 plaques/well for 87% of diverse DENV strains [n = 1521]) and a mode that allows the user to fine-tune the parameters used for counting plaques. We compared standardized manual and Viridot plaque counting methods applied to the same wells by two analyses and found that Viridot plaque counts were as similar to the same analyst's manual count (Lin's concordance correlation coefficient, ρc = 0.99 [95% confidence interval: 0.99-1.00]) as manual counts between analysts (ρc = 0.99 [95% CI: 0.98-0.99]). The average ratio of neutralizing antibody titers based on manual counted plaques to Viridot counted plaques was 1.05 (95% CI: 0.98-1.14), similar to the average ratio of antibody titers based on manual plaque counts by the two analysts (1.06 [95% CI: 0.84-1.34]). Across diverse DENV and ZIKV strains (n = 14), manual and Viridot plaque counts were mostly consistent (range of ρc = 0.74 to 1.00) and the average ratio of antibody titers based on manual and Viridot counted plaques was close to 1 (0.94 [0.86-1.02]). Thus, Viridot can be used for plaque counting and neutralizing antibody titer estimation of diverse DENV strains and potentially other viruses on 96-well plates as well as for formalization of plaque-counting rules for standardization across experiments and analysts

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigen

A genetic locus complements resistance to Bordetella pertussis-induced histamine sensitization.

Histamine plays pivotal role in normal physiology and dysregulated production of histamine or signaling through histamine receptors (HRH) can promote pathology. Previously, we showed that Bordetella pertussis or pertussis toxin can induce histamine sensitization in laboratory inbred mice and is genetically controlled by Hrh1/HRH1. HRH1 allotypes differ at three amino acid residues with

The kinematics and excitation of infrared water vapor emission from planet-forming disks: results from spectrally-resolved surveys and guidelines for JWST spectra

This work presents water emission spectra at wavelengths covered by JWST (2.9-12.8 $\mu$m) as spectrally-resolved with high resolving powers (R = 30,000-100,000) using ground-based spectrographs. Two new surveys with iSHELL and VISIR are combined with previous spectra from CRIRES and TEXES to cover parts of multiple ro-vibrational and rotational bands observable within telluric transmission bands, for a total of 85 disks and $\approx160$ spectra. The general expectation of a range of regions and excitation conditions traced by infrared water spectra is for the first time supported by the combined kinematics and excitation as spectrally resolved at multiple wavelengths. The main findings from this analysis are: 1) water lines are progressively narrower going from the ro-vibrational bands at 2-9 $\mu$m to the rotational lines at 12 $\mu$m, and partly match a broad (BC) and narrow (NC) emission components, respectively, as extracted from ro-vibrational CO spectra; 2) rotation diagrams of resolved water lines from upper level energies of 4000-9500 K show curvatures indicative of optically thick emission ($\approx 10^{18}$ cm$^{-2}$) from a range of excitation temperatures ($\approx$ 800-1100 K); 3) the new 5 $\mu$m spectra demonstrate that slab model fits to the rotational lines at $> 10 \mu$m strongly over-predict the ro-vibrational emission bands at $< 9 \mu$m, implying non-LTE excitation. We discuss these findings in the context of a emission from a disk surface and a molecular inner disk wind, and provide a list of detailed guidelines to support the analysis and interpretation of spectrally-unresolved JWST spectra.Comment: Posted on arXiv as submitted to AJ, for immediate access by teams working on the analysis of JWST spectr

Longitudinal analysis of acute and convalescent B cell responses in a human primary dengue serotype 2 infection model

Background: Acute viral infections induce a rapid and transient increase in antibody-secreting plasmablasts. At convalescence, memory B cells (MBC) and long-lived plasma cells (LLPC) are responsible for long-term humoral immunity. Following an acute viral infection, the specific properties and relationships between antibodies produced by these B cell compartments are poorly understood. Methods:Weutilized a controlled human challenge model of primary dengue virus serotype 2 (DENV2) infection to study acute and convalescent B-cell responses. Findings: The level of DENV2 replication was correlated with the magnitude of the plasmablast response. Functional analysis of plasmablast-derived monoclonal antibodies showed that the DENV2-specific response was dominated by cells producing DENV2 serotype-specific antibodies. DENV2-neutralizing antibodies targeted quaternary structure epitopes centered on domain III of the viral envelope protein (EDIII). Functional analysis ofMBC and serum antibodies from the same subjects six months post-challenge revealed maintenance of the serotypespecific response in both compartments. The serumresponse mainly targeted DENV2 serotype-specific epitopes on EDIII. Interpretation: Our data suggest overall functional alignment of DENV2-specific responses from the plasmablast, through the MBC and LLPC compartments following primary DENV2 inflection. These results provide enhanced resolution of the temporal and specificity of the B cell compartment in viral infection and serve as framework for evaluation of B cell responses in challenge models

Structure and neutralization mechanism of a human antibody targeting a complex Epitope on Zika virus

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency

Catching Element Formation In The Act

Gamma-ray astronomy explores the most energetic photons in nature to address some of the most pressing puzzles in contemporary astrophysics. It encompasses a wide range of objects and phenomena: stars, supernovae, novae, neutron stars, stellar-mass black holes, nucleosynthesis, the interstellar medium, cosmic rays and relativistic-particle acceleration, and the evolution of galaxies. MeV gamma-rays provide a unique probe of nuclear processes in astronomy, directly measuring radioactive decay, nuclear de-excitation, and positron annihilation. The substantial information carried by gamma-ray photons allows us to see deeper into these objects, the bulk of the power is often emitted at gamma-ray energies, and radioactivity provides a natural physical clock that adds unique information. New science will be driven by time-domain population studies at gamma-ray energies. This science is enabled by next-generation gamma-ray instruments with one to two orders of magnitude better sensitivity, larger sky coverage, and faster cadence than all previous gamma-ray instruments. This transformative capability permits: (a) the accurate identification of the gamma-ray emitting objects and correlations with observations taken at other wavelengths and with other messengers; (b) construction of new gamma-ray maps of the Milky Way and other nearby galaxies where extended regions are distinguished from point sources; and (c) considerable serendipitous science of scarce events -- nearby neutron star mergers, for example. Advances in technology push the performance of new gamma-ray instruments to address a wide set of astrophysical questions.Comment: 14 pages including 3 figure

Infant antibody and B-cell responses following confirmed pediatric GII.17 norovirus infections functionally distinguish GII.17 genetic clusters

Genogroup II (GII) noroviruses are a major cause of diarrheal disease burden in children in both high- and low-income countries. GII.17 noroviruses are composed of distinct genetic clusters (I, II, IIIa, and IIIb) and have shown potential for replacing historically more prevalent GII.4 strains, but the serological basis for GII.17 antigenic diversity has not been studied in children. Utilizing samples from a birth cohort, we investigated antibody and B-cell responses to GII.17 cluster variants in confirmed GII.17 infections in young children as well as demonstrated that the distinct genetic clusters co-circulate. Polyclonal serum antibodies bound multiple clusters but showed cluster-specific blockade activity in a surrogate virus neutralization assay. Antibodies secreted by immortalized memory B cells (MBCs) from an infant GII.17 case were highly specific to GII.17 and exhibited blockade activity against this genotype. We isolated an MBC-derived GII.17-specific Immunoglobulin A (IgA) monoclonal antibody called NVA.1 that potently and selectively blocked GII.17 cluster IIIb and recognized an epitope targeted in serum from cluster IIIb–infected children. These data indicate that multiple antigenically distinct GII.17 variants co-circulate in young children, suggesting retention of cluster diversity alongside potential for immune escape given the existence of antibody-defined cluster-specific epitopes elicited during infection

B cell–intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans

Engagement of cytokine receptors by specific ligands activate Janus kinase–signal transducer and activator of transcription (STAT) signaling pathways. The exact roles of STATs in human lymphocyte behavior remain incompletely defined. Interleukin (IL)-21 activates STAT1 and STAT3 and has emerged as a potent regulator of B cell differentiation. We have studied patients with inactivating mutations in STAT1 or STAT3 to dissect their contribution to B cell function in vivo and in response to IL-21 in vitro. STAT3 mutations dramatically reduced the number of functional, antigen (Ag)-specific memory B cells and abolished the ability of IL-21 to induce naive B cells to differentiate into plasma cells (PCs). This resulted from impaired activation of the molecular machinery required for PC generation. In contrast, STAT1 deficiency had no effect on memory B cell formation in vivo or IL-21–induced immunoglobulin secretion in vitro. Thus, STAT3 plays a critical role in generating effector B cells from naive precursors in humans. STAT3-activating cytokines such as IL-21 thus underpin Ag-specific humoral immune responses and provide a mechanism for the functional antibody deficit in STAT3-deficient patients