Characterization of CD8+ T cells in immune-privileged organs of ZIKV-infected Ifnar1-/- mice.

Zika virus (ZIKV) infection caused neurological complications and male infertility, leading to the accumulation of antigen-specific immune cells in immune-privileged organs (IPOs). Thus, it is important to understand the immunological responses to ZIKV in IPOs. We extensively investigated the ZIKV-specific T cell immunity in IPOs in Ifnar1-/- mice, based on an immunodominant epitope E294-302 tetramer. The distinct kinetics and functions of virus-specific CD8+ T cells infiltrated into different IPOs were characterized, with late elevation in the brain and spinal cord. Single epitope E294-302-specific T cells can account for 20-60% of the total CD8+ T cells in the brain, spinal cord, and testicle and persist for at least 90 days in the brain and spinal cord. The E294-302-specific TCRαßs within the IPOs are featured with the majority of clonotypes utilizing TRAV9N-3 paired with diverse TRBV chains, but with distinct αß paired clonotypes in 7 and 30 days post-infection. Specific chemokine receptors, Ccr2 and Ccr5, were selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of virus-specific CD8+ T cells after infection. Overall, this study adds to the understanding of virus-specific CD8+ T cell responses for controlling and clearing ZIKV infection in IPOs.IMPORTANCEThe immune-privileged organs (IPOs), such as the central nervous system and testicles, presented pathogenicity and inflammation after Zika virus (ZIKV) infection with infiltrated CD8+ T cells. Our data show that CD8+ T cells keep up with virus increases and decreases in immune-privileged organs. Furthermore, our study provides the first ex vivo comparative analyses of the composition and diversity related to TCRα/ß clonotypes across anatomical sites and ZIKV infection phases. We show that the vast majority of TCRα/ß clonotypes in tissues utilize TRAV9N-3 with conservation. Specific chemokine expression, including Ccr2 and Ccr5, was found to be selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of the virus-specific CD8+ T cells after the infection. Our study adds insights into the anti-viral immunological characterization and chemotaxis mechanism of virus-specific CD8+ T cells after ZIKV infection in different IPOs.

Defining a de novo non-RBM antibody as RBD-8 and its synergistic rescue of immune-evaded antibodies to neutralize Omicron SARS-CoV-2.

Currently, monoclonal antibodies (MAbs) targeting the SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein are classified into seven classes based on their binding epitopes. However, most of these antibodies are seriously impaired by SARS-CoV-2 Omicron and its subvariants, especially the recent BQ.1.1, XBB and its derivatives. Identification of broadly neutralizing MAbs against currently circulating variants is imperative. In this study, we identified a "breathing" cryptic epitope in the S protein, named as RBD-8. Two human MAbs, BIOLS56 and IMCAS74, were isolated recognizing this epitope with broad neutralization abilities against tested sarbecoviruses, including SARS-CoV, pangolin-origin coronaviruses, and all the SARS-CoV-2 variants tested (Omicron BA.4/BA.5, BQ.1.1, and XBB subvariants). Searching through the literature, some more RBD-8 MAbs were defined. More importantly, BIOLS56 rescues the immune-evaded antibody, RBD-5 MAb IMCAS-L4.65, by making a bispecific MAb, to neutralize BQ.1 and BQ.1.1, thereby producing an MAb to cover all the currently circulating Omicron subvariants. Structural analysis reveals that the neutralization effect of RBD-8 antibodies depends on the extent of epitope exposure, which is affected by the angle of antibody binding and the number of up-RBDs induced by angiotensin-converting enzyme 2 binding. This cryptic epitope which recognizes non- receptor binding motif (non-RBM) provides guidance for the development of universal therapeutic antibodies and vaccines against COVID-19.

Co-exposure to organic UV filters and phthalates and their associations with oxidative stress levels in children: A prospective follow-up study in China.

Children are highly vulnerable to environmental pollutants, especially endocrine-disrupting chemicals (EDCs). Previous research has linked both organic UV filters and phthalates exposure to adiposity and pubertal development in children. Nevertheless, the individual and collective effects of these chemicals on this population remain poorly understood. In this study, twelve organic UV filters and metabolites, six phthalate metabolites and two oxidative stress biomarkers were analyzed in a prospective follow-up study in Shanghai, China after a baseline study conducted 1.5 years earlier. Results revealed a positive association between exposure to individual organic UV filters or their mixture and levels of 8-OHdG (ß ranging from 0.242 to 0.588, P < 0.05), a marker of oxidative DNA damage. BP-3 and OD-PABA made a greater contribution to oxidative DNA damage than other UV filters. Levels of 8-OHdG were also positively correlated with single phthalate metabolites and their mixture, with MnBP and MMP contributing the most. Stratified analysis found that these associations were mainly observed in girls. Our mixture analysis revealed cumulative risks of oxidative DNA damage when there was co-exposure to these two kinds of EDCs. These results underscore the importance of considering the risks associated with organic UV filters and the necessity of evaluating the effects of all these pollutants, both individually and in mixtures.

Chlorination of bisphenols in water: Understanding the kinetics and formation mechanism of 2-butene-1,4-dial and analogues.

While it is widely accepted that 2-butene-1,4-dial (BDA) is a toxic metabolite with genotoxic and carcinogenic properties, little is known about BDA and its analogues (BDAs) formation during water disinfection. In this study, the effects of different chlorination conditions on the formation of BDAs from bisphenol and its analogues (BPs analogues) were evaluated. A transformation pathway for the formation of BDAs upon chlorination of BPs analogues is proposed. The time profile of the transformation of BPs analogues into BDAs reveals that the generation of dichlorohydroquinone, dichloro-hydroxybenzenesulfonic acid and 2,4,6-trichlorophenol, are significantly associated with the formation of BDAs in the disinfected water. Owing to the different bridging groups contributing to the electrophilicity of BPs analogues in varying degrees, the stronger the electrophilicity of BPs analogues the more BDAs are formed. In addition, the type of BDAs produced is also affected. Four types of BDAs were detected in this study, one of which was newly identified. This study confirms that BPs analogues are an important source of BDAs and provides more insights into the formation of BDAs during chlorination. Greater attention should be given to the formation of BDAs in chlorinated water and their potential threat to humans and the ecosystem.

Total Luminescence Spectroscopy for Quantification of Temperature Effects on Photophysical Properties of Photoluminescent Materials.

Quantification of the temperature effects on the optical properties of photoluminescent (PL) materials is important for a fundamental understanding of both materials optical processes and rational PL materials design and applications. However, existing techniques for studying the temperature effects are limited in their information content. Reported herein is a temperature-dependent total photoluminescence (TPL) spectroscopy technique for probing the temperature dependence of materials optical properties. When used in combination with UV-vis measurements, this TPL method enables experimental quantification of temperature effects on fluorophore fluorescence intensity and quantum yield at any combination of excitation and detection wavelengths, including the fluorophore Stokes-shifted and anti-Stokes-shifted fluorescence. All model polyaromatic hydrocarbon (PAH) and xanthene fluorophores exhibited a strong excitation- and emission-wavelength dependence in their temperature effects. However, the heavy-atom effects used for explaining the strong temperature dependence of brominated anthracenes are not operative with xanthene fluorophores that have heavy atom substitutions. The insights from TPL measurements are important not only for enhancing the fundamental understandings of the materials photophysical properties but also for rational measurement design for applications where the temperature sensitivity of the fluorophore fluorescence is critical. An example application is demonstrated for developing a sensitive and robust ratiometric fluorescence thermometric method for in situ real-time monitoring of sample temperatures inside a fluorescence cuvette placed in a temperature-controlled sample holder.

A Wider and Deeper Peptide-Binding Groove for the Class I Molecules from B15 Compared with B19 Chickens Correlates with Relative Resistance to Marek's Disease.

The chicken MHC is known to confer decisive resistance or susceptibility to various economically important pathogens, including the iconic oncogenic herpesvirus that causes Marek's disease (MD). Only one classical class I gene, BF2, is expressed at a high level in chickens, so it was relatively easy to discern a hierarchy from well-expressed thermostable fastidious specialist alleles to promiscuous generalist alleles that are less stable and expressed less on the cell surface. The class I molecule BF2*1901 is better expressed and more thermostable than the closely related BF2*1501, but the peptide motif was not simpler as expected. In this study, we confirm for newly developed chicken lines that the chicken MHC haplotype B15 confers resistance to MD compared with B19. Using gas phase sequencing and immunopeptidomics, we find that BF2*1901 binds a greater variety of amino acids in some anchor positions than does BF2*1501. However, by x-ray crystallography, we find that the peptide-binding groove of BF2*1901 is narrower and shallower. Although the self-peptides that bound to BF2*1901 may appear more various than those of BF2*1501, the structures show that the wider and deeper peptide-binding groove of BF2*1501 allows stronger binding and thus more peptides overall, correlating with the expected hierarchies for expression level, thermostability, and MD resistance. Our study provides a reasonable explanation for greater promiscuity for BF2*1501 compared with BF2*1901, corresponding to the difference in resistance to MD.

The CD8+ and CD4+ T Cell Immunogen Atlas of Zika Virus Reveals E, NS1 and NS4 Proteins as the Vaccine Targets.

Zika virus (ZIKV)-specific T cells are activated by different peptides derived from virus structural and nonstructural proteins, and contributed to the viral clearance or protective immunity. Herein, we have depicted the profile of CD8+ and CD4+ T cell immunogenicity of ZIKV proteins in C57BL/6 (H-2b) and BALB/c (H-2d) mice, and found that featured cellular immunity antigens were variant among different murine alleles. In H-2b mice, the proteins E, NS2, NS3 and NS5 are recognized as immunodominant antigens by CD8+ T cells, while NS4 is dominantly recognized by CD4+ T cells. In contrast, in H-2d mice, NS1 and NS4 are the dominant CD8+ T cell antigen and NS4 as the dominant CD4+ T cell antigen, respectively. Among the synthesized 364 overlapping polypeptides spanning the whole proteome of ZIKV, we mapped 91 and 39 polypeptides which can induce ZIKV-specific T cell responses in H-2b and H-2d mice, respectively. Through the identification of CD8+ T cell epitopes, we found that immunodominant regions E294-302 and NS42351-2360 are hotspots epitopes with a distinct immunodominance hierarchy present in H-2b and H-2d mice, respectively. Our data characterized an overall landscape of the immunogenic spectrum of the ZIKV polyprotein, and provide useful insight into the vaccine development.

Structural and inhibitor sensitivity analysis of influenza B-like viral neuraminidases derived from Asiatic toad and spiny eel.

Influenza virus neuraminidase (NA) is an important target for antiviral development because it plays a crucial role in releasing newly assembled viruses. Two unique influenza-like virus genomes were recently reported in the Wuhan Asiatic toad and Wuhan spiny eel. Their NA genes appear to be highly divergent from all known influenza NAs, raising key questions as to whether the Asiatic toad influenza-like virus NA (tNA) and spiny eel NA (eNA) have canonical NA activities and structures and whether they show sensitivity to NA inhibitors (NAIs). Here, we found that both tNA and eNA have neuraminidase activities. A detailed structural analysis revealed that tNA and eNA present similar overall structures to currently known NAs, with a conserved calcium binding site. Inhibition assays indicated that tNA is resistant to NAIs, while eNA is still sensitive to NAIs. E119 is conserved in canonical NAs. The P119E substitution in tNA can restore sensitivity to NAIs, and, in contrast, the E119P substitution in eNA decreased its sensitivity to NAIs. The structures of NA-inhibitor complexes further provide a detailed insight into NA-inhibitor interactions at the atomic level. Moreover, tNA and eNA have unique N-glycosylation sites compared with canonical NAs. Collectively, the structural features, NA activities, and sensitivities to NAIs suggest that fish- and amphibian-derived influenza-like viruses may circulate in these vertebrates. More attention should be paid to these influenza-like viruses because their NA molecules may play roles in the emergence of NAI resistance.

Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease.

Oridonin Inhibits SARS-CoV-2 Oridonin, a natural product extracted from Rabdosia rubescens, possesses a wide range of pharmacological properties, including anti-inflammatory, anti-cancer, anti-microbial, neuroprotection, immunoregulation, etc. In article number 2100124, Baisen Zhong, Litao Sun, and co-workers demonstrate that Oridonin targets the SARS-CoV-2 3CL protease by covalently binding to cysteine145 in its active pocket to exert an anti-SARS-CoV-2 effect, which provides a novel candidate for the treatment of COVID-19.

Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease.

The current COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an enormous threat to public health. The SARS-CoV-2 3C-like protease (3CLpro), which is critical for viral replication and transcription, has been recognized as an ideal drug target. Herein, it is identified that three herbal compounds, Salvianolic acid A (SAA), (-)-Epigallocatechin gallate (EGCG), and Oridonin, directly inhibit the activity of SARS-CoV-2 3CLpro. Further, blocking SARS-CoV-2 infectivity by Oridonin is confirmed in cell-based experiments. By solving the crystal structure of 3CLpro in complex with Oridonin and comparing it to that of other ligands with 3CLpro, it is identified that Oridonin binds at the 3CLpro catalytic site by forming a C-S covalent bond, which is confirmed by mass spectrometry and kinetic study, blocking substrate binding through a nonpeptidomimetic covalent binding mode. Thus, Oridonin is a novel candidate to develop a new antiviral treatment for COVID-19.

An engineered bispecific human monoclonal antibody against SARS-CoV-2.

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19.

Kinetic spectroscopic quantification using two-step chromogenic and fluorogenic reactions: From theoretical modeling to experimental quantification of biomarkers in practical samples.

Kinetic chromogenic (CG) and fluorogenic (FG) quantification deduces analyte concentration based on the reaction rate between the CG/FG probe and its targeted molecule. Little progress has been made in the past half century in either the theory or the applications of the kinetic spectroscopic quantification methods. Current kinetic CG/FG quantification is limited only to a subset of CG/FG reactions that can be approximated as the single-step process, and more problematically, to research samples with no matrix interferences. Reported herein is a kinetic quantification model established for multistep CG/FG reactions and a proof-of-concept demonstration of direct kinetic FG quantification of biomarkers in practical samples. The kinetic spectral intensity of the CG/FG reactions with two rate-limiting steps comprises three temporal regions: an accelerating period where rate of signal change is increasingly rapid, a linear region where the rate of signal change is approximately constant, and a deceleration region where the rate of signal increase becomes progressively small. Kinetic quantification is performed through simple linear-curve-fitting of the kinetic signal in its linear time-course region. The theoretical model is validated with the dual CG/FG 2-thiobarbituric acid (TBA) and malondialdehyde (MDA) reaction. Proof-of-concept kinetic spectroscopic quantification of analytes in practical samples is demonstrated with the FG quantification of MDA in canned chicken. The only sample preparation is bench-top centrifugation followed by two sequential syringe filtrations. The total kinetic FG assay time is less than 10 min, more than 10 times more efficient than the current equilibrium-based MDA assay. The theoretical model and the measurement design strategies offered by this work should help transform the current kinetic spectroscopic quantification from a niche research tool to an indispensable technique for time-sensitive applications.

Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus.

Here, we investigate a monoclonal antibody, Z2B3, isolated from an H7N9-infected patient, that exhibited cross-reactivity to both N9 (group 2) and a broad range of seasonal and avian N1 (group 1) proteins but lost activity to the N1 with the substitution K432E. This substitution exists in 99.25% of seasonal influenza strains after 2013. The NA-Z2B3 complex structures indicated that Z2B3 binds within the conserved active site of the neuraminidase (NA) protein. A salt bridge between D102 in Z2B3 and K432 in NA plays an important role in binding. Structure-based modification of Z2B3 with D102R in heavy chain reversed the salt bridge and restored the binding and inhibition of N1 with E432. Furthermore, Z2B3-D102R can protect mice from A/Serbia/NS-601/2014 H1N1 virus (NA contains E432) infection while the wild-type Z2B3 antibody shows no protection. This study demonstrates that a broadly reactive and protective antibody to NA can be in principle edited to restore binding and inhibition to recently drifted N1 NA and regain protection against the variant influenza strain.IMPORTANCE The immune system produces antibodies to protect the human body from harmful invaders. The monoclonal antibody (MAb) is one kind of effective antivirals. In this study, we isolated an antibody (Z2B3) from an H7N9 influenza virus-infected child. It shows cross-reactivity to both group 1 (N1) and group 2 (N9) neuraminidases (NAs) but is sensitive to N1 NA with a K432E substitution. Structural analysis of the NA-antibody fragment antigen-binding (Fab) complex provides a clue for antibody modification, and the modified antibody restored binding and inhibition to recently drifted N1 NA and regained protection against the variant influenza strain. This finding suggests that antibodies to NA may be a useful therapy and can be in principle edited to defeat drifted influenza virus.

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2.

Neutralizing antibodies could potentially be used as antivirals against the coronavirus disease 2019 (COVID-19) pandemic. Here, we report isolation of four human-origin monoclonal antibodies from a convalescent patient, all of which display neutralization abilities. The antibodies B38 and H4 block binding between the spike glycoprotein receptor binding domain (RBD) of the virus and the cellular receptor angiotensin-converting enzyme 2 (ACE2). A competition assay indicated different epitopes on the RBD for these two antibodies, making them a potentially promising virus-targeting monoclonal antibody pair for avoiding immune escape in future clinical applications. Moreover, a therapeutic study in a mouse model validated that these antibodies can reduce virus titers in infected lungs. The RBD-B38 complex structure revealed that most residues on the epitope overlap with the RBD-ACE2 binding interface, explaining the blocking effect and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide a structural basis for rational vaccine design.

Salt bridge-forming residues positioned over viral peptides presented by MHC class I impacts T-cell recognition in a binding-dependent manner.

The viral peptides presentation by major histocompatibility complex class I (MHC I) molecules play a pivotal role in T-cell recognition and the subsequent virus clearance. This process is delicately adjusted by the variant residues of MHC I, especially the residues in the peptide binding groove (PBG). In a series of MHC I molecules, a salt bridge is formed above the N-terminus of the peptides. However, the potential impact of the salt bridge on peptide binding and T-cell receptor (TCR) recognition of MHC I, as well as the corresponding molecular basis, are still largely unknown. Herein, we determined the structures of HLA-B*4001 and H-2Kd in which two different types of salt bridges (Arg62-Glu163 or Arg66-Glu163) across the PBG were observed. Although the two salt bridges led to different conformation shifts of both the MHC I α helix and the peptides, binding of the peptides with the salt bridge residues was relatively conserved. Furthermore, through a series of in vitro and in vivo investigations, we found that MHC I mutations that disrupt the salt bridge alleviate peptide binding and can weaken the TCR recognition of MHC I-peptide complexes. Our study may provide key references for understanding MHC I-restricted peptide recognition by T-cells.

An Invariant Arginine in Common with MHC Class II Allows Extension at the C-Terminal End of Peptides Bound to Chicken MHC Class I.

MHC molecules are found in all jawed vertebrates and are known to present peptides to T lymphocytes. In mammals, peptides can hang out either end of the peptide-binding groove of classical class II molecules, whereas the N and C termini of peptides are typically tightly bound to specific pockets in classical class I molecules. The chicken MHC, like many nonmammalian vertebrates, has a single dominantly expressed classical class I molecule encoded by the BF2 locus. We determined the structures of BF2*1201 bound to two peptides and found that the C terminus of one peptide hangs outside of the groove with a conformation much like the peptides bound to class II molecules. We found that BF2*1201 binds many peptides that hang out of the groove at the C terminus, and the sequences and structures of this MHC class I allele were determined to investigate the basis for this phenomenon. The classical class I molecules of mammals have a nearly invariant Tyr (Tyr84 in humans) that coordinates the peptide C terminus, but all classical class I molecules outside of mammals have an Arg in that position in common with mammalian class II molecules. We find that this invariant Arg residue switches conformation to allow peptides to hang out of the groove of BF2*1201, suggesting that this phenomenon is common in chickens and other nonmammalian vertebrates, perhaps allowing the single dominantly expressed class I molecule to bind a larger repertoire of peptides.