Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity.

A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19.

Vitamin D kinetics in nonpregnant and pregnant women after a single oral dose of trideuterated vitamin D3.

The plasma pool of the hormone 1,25-dihydroxyvitamin D (1,25(OH)2D) is increased throughout most of human pregnancy. Mechanisms behind this adaptation are unclear, in part due to limited data on vitamin D kinetics during pregnancy. Stable isotopes make it possible to study vitamin D kinetics in vulnerable study populations like pregnant women. We conducted a pilot study of vitamin D kinetics in nonpregnant and pregnant women. We evaluated a clinical protocol and developed analytical methods to assess the serum appearance and disappearance of trideuterated vitamin D3 (d3-vitamin D3) and trideuterated 25-hydroxyvitamin D3 (d3-25(OH)D3) after a single oral dose of 25 µg of [6,19,19-2H]-vitamin D3 (d3-vitamin D3). Blood was collected at baseline and 2, 4, 6, 24, 168, 264, and 456 hours post-dosing. We then described the serum kinetic profiles of d3-vitamin D3 and d3-25(OH)D3 in nonpregnant and pregnant women. Serum kinetic profiles of d3-vitamin D3 and d3-25(OH)D3 followed a time course in line with previous pharmacokinetic studies. There was marked variability between participants in the area under the concentration-time curve (AUC) of d3-25(OH)D3 over the 20-day study period. This AUC of d3-25(OH)D3 was positively correlated with the serum vitamin D binding protein (DBP) concentration, which was higher in pregnant compared with nonpregnant women. The mean serum half-life of 25(OH)D3 was longer but not significantly different in pregnant women (18.8 days) compared with nonpregnant women (13.6 days). Our pilot study demonstrated that a single oral dose of 25 µg of d3-vitamin D3 can be used to study vitamin D kinetics. Serum DBP concentration is an important predictor of vitamin D kinetics, and more research is needed to fully understand the significance of elevated DBP concentration during pregnancy.

Structurally and functionally distinct early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response.

A damaging inflammatory response is strongly implicated in the pathogenesis of severe COVID-19 but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated, anti-SARS-CoV-2 IgG predicted progression from mild, to more severe COVID-19. In contrast to the antibody structures that predicted disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were low in Fc afucosylation and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model which revealed that human IgG-FcγR interactions can regulate inflammation in the lung. Afucosylated IgG immune complexes induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine elicited IgG did not promote an inflammatory lung response. Here, we show that IgG-FcγR interactions can regulate inflammation in the lung and define distinct lung activities associated with the IgG that predict severe COVID-19 and protection against SARS-CoV-2. ONE SENTENCE SUMMARY: Divergent early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response and are functionally distinct in vivo .

Proinflammatory IgG Fc structures in patients with severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 infections can cause coronavirus disease 2019 (COVID-19), which manifests with a range of severities from mild illness to life-threatening pneumonia and multi-organ failure. Severe COVID-19 is characterized by an inflammatory signature, including high levels of inflammatory cytokines, alveolar inflammatory infiltrates and vascular microthrombi. Here we show that patients with severe COVID-19 produced a unique serologic signature, including an increased likelihood of IgG1 with afucosylated Fc glycans. This Fc modification on severe acute respiratory syndrome coronavirus 2 IgGs enhanced interactions with the activating Fcγ receptor FcγRIIIa; when incorporated into immune complexes, Fc afucosylation enhanced production of inflammatory cytokines by monocytes, including interleukin-6 and tumor necrosis factor. These results show that disease severity in COVID-19 correlates with the presence of proinflammatory IgG Fc structures, including afucosylated IgG1.

Comparison of MS2, synchronous precursor selection MS3, and real-time search MS3 methodologies for lung proteomes of hydrogen sulfide treated swine.

Tandem mass tags (TMTs) have increasingly become an attractive technique for global proteomics. However, its effectiveness for multiplexed quantitation by traditional tandem mass spectrometry (MS2) suffers from ratio distortion. Synchronous precursor selection (SPS) MS3 has been widely accepted for improved quantitation accuracy, but concurrently decreased proteome coverage. Recently, a Real-Time Search algorithm has been integrated with the SPS MS3 pipeline (RTS MS3) to provide accurate quantitation and improved depth of coverage. In this mechanistic study of the impact of exposure to hydrogen sulfide (H2S) on the respiration of swine, we used TMT-based comparative proteomics of lung tissues from control and H2S-treated subjects as a test case to evaluate traditional MS2, SPS MS3, and RTS MS3 acquisition methods on both the Orbitrap Fusion and Orbitrap Eclipse platforms. Comparison of the results obtained by the MS2 with those of SPS MS3 and RTS MS3 methods suggests that the MS3-driven quantitative strategies provided a more accurate global-scale quantitation; however, only RTS MS3 provided proteomic coverage that rivaled that of traditional MS2 analysis. RTS MS3 not only yields more productive MS3 spectra than SPS MS3 but also appears to focus the analysis more effectively on unique peptides. Furthermore, pathway enrichment analyses of the H2S-altered proteins demonstrated that an additional apoptosis pathway was discovered exclusively by RTS MS3. This finding was verified by RT-qPCR, western blotting, and TUNEL staining experiments. We conclude that RTS MS3 workflow enables simultaneous improvement of quantitative accuracy and proteome coverage over alternative approaches (MS2 and SPS MS3). Graphical abstract.

Proinflammatory IgG Fc structures in patients with severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections can cause Coronavirus Disease 2019 (COVID-19), which manifests with a range of severities from mild illness to life threatening pneumonia and multi-organ failure. Severe COVID-19 is characterized by an inflammatory signature including high levels of inflammatory cytokines, alveolar inflammatory infiltrates and vascular microthrombi. Here we show that severe COVID-19 patients produced a unique serologic signature, including increased IgG1 with afucosylated Fc glycans. This Fc modification on SARS-CoV-2 IgGs enhanced interactions with the activating FcγR, FcγRIIIa; when incorporated into immune complexes, Fc afucosylation enhanced production of inflammatory cytokines by monocytes, including IL-6 and TNF. These results show that disease severity in COVID-19 correlates with the presence of afucosylated IgG1, a pro-inflammatory IgG Fc modification.

Maternal Anti-Dengue IgG Fucosylation Predicts Susceptibility to Dengue Disease in Infants.

Infant mortality from dengue disease is a devastating global health burden that could be minimized with the ability to identify susceptibility for severe disease prior to infection. Although most primary infant dengue infections are asymptomatic, maternally derived anti-dengue immunoglobulin G (IgGs) present during infection can trigger progression to severe disease through antibody-dependent enhancement mechanisms. Importantly, specific characteristics of maternal IgGs that herald progression to severe infant dengue are unknown. Here, we define ≥10% afucosylation of maternal anti-dengue IgGs as a risk factor for susceptibility of infants to symptomatic dengue infections. Mechanistic experiments show that afucosylation of anti-dengue IgGs promotes FcγRIIIa signaling during infection, in turn enhancing dengue virus replication in FcγRIIIa+ monocytes. These studies identify a post-translational modification of anti-dengue IgGs that correlates with risk for symptomatic infant dengue infections and define a mechanism by which afucosylated antibodies and FcγRIIIa enhance dengue infections.

FcRn, but not FcγRs, drives maternal-fetal transplacental transport of human IgG antibodies.

The IgG Fc domain has the capacity to interact with diverse types of receptors, including the neonatal Fc receptor (FcRn) and Fcγ receptors (FcγRs), which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis, are mediated by FcγRs, which upon cross-linking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and nonoverlapping functional properties of FcRn and FcγRs, recent studies have suggested that FcγRs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of FcγRs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter FcγRIIIa or FcRn binding, half-life, or their ability to deplete target cells in FcγR/FcRn humanized mice. Modeling maternal-fetal transport in FcγR/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing FcγRIIIa binding did not result in enhanced maternal-fetal transport. These results argue against a role for FcγRs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to enhance only FcRn binding as a means to improve maternal-fetal transport of IgG.

Serum Proteomics on the Basis of Discovery of Predictive Biomarkers of Response to Androgen Deprivation Therapy in Advanced Prostate Cancer.

BACKGROUND: We investigated the serum proteome of hormone-sensitive prostate cancer patients to determine candidate biomarkers associated with androgen deprivation therapy (ADT) efficacy. PATIENTS AND METHODS: Serum proteomes generated using isobaric mass tags for relative and absolute quantitation were analyzed using reverse-phase liquid chromatography coupled to tandem mass spectrometry. The advanced hormone-sensitive prostate cancer cohorts studied were: (1) untreated "paired" pre-ADT and 4-month post-ADT hormone-sensitive patients (n = 15); (2) "early ADT failure" patients (n = 10) in whom ADT treatment failed within a short period of time; and (3) "late ADT failure" patients (n = 10) in whom ADT treatment failed after a prolonged response time. Differential abundance was assessed, and ingenuity pathway analysis (IPA) was used to identify interaction networks in selected candidates from these comparisons. RESULTS: Between "post-ADT" and combined "early" and "late" ADT failure groups 149 differentially detected candidates were observed, and between "early" and "late" ADT failure groups 98 candidates were observed; 47 candidates were common in both comparisons. IPA network enrichment analysis of the 47 candidates identified 3 interaction networks (P < .01) including 17-ß-estradiol, nuclear factor kappa-light-chain enhancer of activated B cells complex, and P38 mitogen-activated protein kinases as pathways with potential markers of response to ADT. CONCLUSION: A global proteomic analysis identified pathways with markers of ADT response, which will need validation in independent data sets.

Time Transfer by Laser Link (T2L2) in Noncommon View Between Europe and China.

The Time Transfer by Laser Link (T2L2) project allows for the synchronization of remote ultrastable clocks over intercontinental distances. The principle is derived from the satellite laser ranging technology with a dedicated space equipment designed to record arrival times of laser pulses at the satellite. The space segment was launched in 2008 as a passenger instrument on the ocean altimetry satellite Jason 2. For the first time, we have conducted by the end of 2016 a dedicated time transfer comparison campaign between Global Positioning System and T2L2 over intercontinental distances. The campaign was carried out between two laboratories in Europe and two in China. The campaign has demonstrated a consistency of the time transfer techniques at the 1-ns level, together with the confirmation of a subnanosecond level for continental distances.

Physiological and proteomic analyses of Fe(III)-reducing co-cultures of Desulfotomaculum reducens MI-1 and Geobacter sulfurreducens PCA.

We established Fe(III)-reducing co-cultures of two species of metal-reducing bacteria, the Gram-positive Desulfotomaculum reducens MI-1 and the Gram-negative Geobacter sulfurreducens PCA. Co-cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens relied upon products of pyruvate oxidation by D. reducens (acetate, hydrogen) for use as electron donor in the co-culture. Co-cultures reduced Fe(III) to Fe(II) robustly, and Fe(II) was consistently detected earlier in co-cultures than pure cultures. Notably, faster cell growth, and correspondingly faster pyruvate oxidation, was observed by D. reducens in co-cultures. Global comparative proteomic analysis was performed to observe differential protein abundance during co-culture vs. pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c-type cytochromes and type IV pili proteins, were significantly increased in abundance in co-cultures relative to pure cultures. D. reducens ribosomal proteins were significantly increased in co-cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co-culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co-cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate significantly.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.

Plazomicin is effective in a non-human primate pneumonic plague model.

The efficacy of plazomicin for pneumonic plague was evaluated in a non-human primate model. African Green monkeys challenged with a lethal aerosol of Yersinia pestis [median (range) of 98 (15-331) LD50s] received placebo (n=12) or 'humanized' dose regimens (6.25, 12.5 or 25mg/kg every 24h) of plazomicin (n=52) after the onset of fever for a duration of 5 or 10days. All animals treated with placebo died, while 36 plazomicin-treated animals survived through study end. The majority (33/36) were either in the 10-day (high-/mid-/low-dose) or 5-day high-dose groups. The findings suggest an exposure range of plazomicin for treatment of pneumonic/bacteremic Y. pestis infection in humans.

Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor.

We describe here the use of label-free wide selected-ion monitoring data-independent acquisition (WiSIM-DIA) to identify proteins that are involved in the formation of tomato (Solanum lycopersicum) fruit cuticles and that are regulated by the transcription factor CUTIN DEFICIENT2 (CD2). A spectral library consisting of 11 753 unique peptides, corresponding to 2338 tomato protein groups, was used and the DIA analysis was performed at the MS1 level utilizing narrow mass windows for extraction with Skyline 2.6 software. We identified a total of 1140 proteins, 67 of which had expression levels that differed significantly between the cd2 tomato mutant and the wild-type cultivar M82. Differentially expressed proteins including a key protein involved in cutin biosynthesis, were selected for validation by target SRM/MRM and by Western blot analysis. In addition to confirming a role for CD2 in regulating cuticle formation, the results also revealed that CD2 influences pathways associated with cell wall biology, anthocyanin biosynthesis, plant development, and responses to stress, which complements findings of earlier RNA-Seq experiments. Our results provide new insights into molecular processes and aspects of fruit biology associated with CD2 function, and demonstrate that the WiSIM-DIA is an effective quantitative approach for global protein identifications.

Pathogenesis-Related Proteins Limit the Retention of Condensed Tannin Additions to Red Wines.

Exogenous additions of condensed tannin (CT) to must or wine are a common winemaking practice, but many studies have reported inexplicably low and variable retention of added CT. We observed that additions of purified CT to red wines can result in the formation of an insoluble precipitate with high nitrogen content. Proteomic analysis of the precipitant identified several classes of pathogenesis-related proteins. Proteins in juices and red wines were quantitated by SDS-PAGE and were highest in native Vitis spp., followed by interspecific hybrids and Vitis vinifera. Wine protein was positively correlated with the ratio of juice protein to the quantity of tannin derived from fruit. The binding of added CT by wine protein could be well modeled by the Freundlich equation. These observations may explain the poor CT retention in previous studies, particularly for interspecific hybrids, and also indicate that protein removal during winemaking may improve exogenous CT retention.

Performance evaluation of Puritan® universal transport system (UniTranz-RT™) for preservation and transport of clinical viruses.

The ability of a non-propagating microbial transport medium to maintain the viability of clinically relevant viruses was compared to a similar commercial medium to establish performance equivalence. Two dilutions of stock of test viruses, namely adenovirus (AdV), cytomegalovirus (CMV), echovirus Type 30 (EV), herpes simplex virus (HSV) types 1 and 2, influenza A, parainfluenza 3 (PIV), respiratory syncytial virus (RSV), and varicella zoster virus (VZV), were spiked into Puritan® Medical Products Company Universal Transport System (UniTranz-RT™) and BD(TM) Universal Viral Transport System (UVT) and incubated at 4 °C and room temperature (RT) for up to 72 hr. Post incubation assessment of recovery of AdV, EV, HSV-2, PIV, and VZV from UniTranz-RT™ and UVT using shell vial assays followed by immunofluorescence staining demonstrated statistically significant differences between both transport media. In general, significantly higher recoveries of AdV, EV, and VZV were found from UniTranz-RT™ than UVT whereas HSV-2 and PIV were recovered better from UVT than UniTranz-RT™, under specific test conditions. The recovery of HSV-1, influenza A, PIV, and RSV showed no significant differences between transport media. Sulforhodamine B-based assay analysis of UniTranz-RT™ lots prior to and at expiration exhibited no cytotoxicity. The overall results of the study validate the full performance of UniTranz-RT™ as a viral transport medium and establish its effectiveness on par with the UVT.

Performance evaluation of two microbial transport media designed for preservation and transport of Chlamydiae, Mycoplasma and Ureaplasma.

The ability of a non-propagating transport device (test device) to maintain the viability of clinically relevant bacteria was compared with a similar commercial device (predicate device) to establish performance equivalence. Test bacteria, namely Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma hominis, Mycoplasma pneumoniae and Ureaplasma urealyticum, were inoculated into the test [Puritan Medical Products Universal Transport System (UniTranz-RT(TM))] and predicate (BD Universal Viral Transport System) devices, and incubated at 4 °C and room temperature for up to 72 h. Bacterial viability was assessed at selected time points post-incubation using shell vial assays followed by immunofluorescence staining (for Chlamydia) or by standard culture techniques (for Mycoplasma and Ureaplasma). Results indicated that the Chlamydia strains were equally stable in both test and predicate devices through 72 h storage, at both test temperatures. Quantifiable levels of Mycoplasma and Ureaplasma were recovered from the test and predicate devices throughout the storage period. Low-temperature storage improved bacterial viability when compared with room temperature storage. In addition, the predicate device demonstrated slightly improved performance versus the test device in the context of Mycoplasma and Ureaplasma following 72 h storage. The overall results of the study confirmed the full performance of UniTranz-RT(TM) as a microbial transport medium and established equal performance with the predicate device.

Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens†MI-1 using an NADH-based activity assay.

Understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. Using non-denaturing separations and mass spectrometry identification, in combination with a colorimetric screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins from the D. reducens proteome not previously characterized as iron reductases. Their function was confirmed by heterologous expression in Escherichia coli. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. The proteins identified are NADH : flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase flavin adenine dinucleotide/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble protein fraction, suggesting a type of membrane association, although PSORTb predicts both proteins are cytoplasmic. This study is the first functional proteomic analysis of D. reducens and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.

Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.

Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.