A new multiplex SARS-CoV-2 antigen microarray showed correlation of IgG, IgA, and IgM antibodies from patients with COVID-19 disease severity and maintenance of relative IgA and IgM antigen binding over time

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity different from the same isotype in whole serum, probably due to competition from the other isotypes present. Using purified antibody isotypes from naïve Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with binding to the S region S1 expressed in insect cells (S1 Sf21) significant for IgG, IgA, and IgM. Assessing longitudinal response for constant concentrations of purified antibody isotypes for a patient subset demonstrated that the relative proportion of antigen-specific IgGs decreased over time for severe disease, but the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen-specific serum IgA and IgM playing a role in maintaining longer-term protection, important for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions

Improved diagnosis of SARS-CoV-2 by using nucleoprotein and spike protein fragment 2 in quantitative dual ELISA tests

The novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative enzyme-linked immunosorbent assays (ELISAs) that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive coronavirus disease-2019 (COVID-19) cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients who had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus

Context-dependency in the effects of nutrient loading and consumers on the availability of space in marine rocky environments

Background: Enhanced nutrient loading and depletion of consumer populations interact to alter the structure of aquatic plant communities. Nonetheless, variation between adjacent habitats in the relative strength of bottom-up (i.e. nutrients) versus top-down (i.e. grazing) forces as determinants of community structure across broad spatial scales remains unexplored. We experimentally assessed the importance of grazing pressure and nutrient availability on the development of macroalgal assemblages and the maintenance of unoccupied space in habitats differing in physical conditions (i.e. intertidal versus subtidal), across regions of contrasting productivity (oligotrophic coasts of South Australia versus the more productive coasts of Eastern Australia). Methodology/Principal findings: In Eastern Australia, grazers were effective in maintaining space free of macroalgae in both intertidal and subtidal habitats, irrespective of nutrient levels. Conversely, in South Australia, grazers could not prevent colonization of space by turf-forming macroalgae in subtidal habitats regardless of nutrients levels, yet in intertidal habitats removal of grazers reduced unoccupied space when nutrients were elevated. Conclusions/Significance: Assessing the effects of eutrophication in coastal waters requires balancing our understanding between local consumer pressure and background oceanographic conditions that affect productivity. This broader-based understanding may assist in reconciling disproportionately large local-scale variation, a characteristic of ecology, with regional scale processes that are often of greater relevance to policy making and tractability to management.Fabio Bulleri, Bayden D. Russell, Sean D. Connel

Diffuse and specific tectopulvinar terminals in the tree shrew: synapses, synapsins, and synaptic potentials.

The pulvinar nucleus of the tree shrew receives both topographic (specific) and nontopographic (diffuse) projections from superior colliculus (SC), which form distinct synaptic arrangements. We characterized the physiological properties of these synapses and describe two distinct types of excitatory postsynaptic potentials (EPSPs) that correlate with structural properties of the specific and diffuse terminals. Synapses formed by specific terminals were found to be significantly longer than those formed by diffuse terminals. Stimulation of these two terminal types elicited two types of EPSPs that differed in their latency and threshold amplitudes. In addition, in response to repetitive stimulation (0.5-20 Hz) one type of EPSP displayed frequency-dependent depression whereas the amplitudes of the second type of EPSP were not changed by repetitive stimulation of up to 20 Hz. To relate these features to vesicle release, we compared the synapsin content of terminals in the pulvinar nucleus and the dorsal lateral geniculate (dLGN) by combining immunohistochemical staining for synapsin I or II with staining for the type 1 or type 2 vesicular glutamate transporters (markers for corticothalamic and tectothalamic/retinogeniculate terminals, respectively). We found that retinogeniculate terminals do not contain either synapsin I or synapsin II, corticothalamic terminals in the dLGN and pulvinar contain synapsin I, but not synapsin II, whereas tectopulvinar terminals contain both synapsin I and synapsin II. Finally, both types of EPSPs showed a graded increase in amplitude with increasing stimulation intensity, suggesting convergence; this was confirmed using a combination of anterograde tract tracing and immunocytochemistry. We suggest that the convergent synaptic arrangements, as well as the unique synapsin content of tectopulvinar terminals, allow them to relay a dynamic range of visual signals from the SC

Frequency-Dependent Release of Substance P Mediates Heterosynaptic Potentiation of Glutamatergic Synaptic Responses in the Rat Visual Thalamus

To investigate the interaction between peptides and glutamatergic synapses in the dorsal thalamus, we compared the frequency-dependent plasticity of excitatory postsynaptic potentials (EPSPs) in the tectorecipient zone of rodent lateral posterior nucleus (LPN), which is densely innervated by axons that contain the neuromodulator substance P (SP). Immunocytochemistry and confocal and electron microscopy revealed that neurokinin 1 (NK1) receptors are distributed on the dendrites of LPN cells, whereas SP is contained in axons originating from the superior colliculus (SC) and is reduced following SC lesions. In vitro whole cell recordings in parasagittal slices revealed that stimulation of the SC or optic radiations (corticothalamic axons [CTXs]) evoked LPN EPSPs that increased in amplitude with increasing stimulation intensity, suggesting convergence. With 0.5- to 10-Hz stimulus trains, CTX EPSP amplitudes displayed frequency-dependent facilitation, whereas SC EPSP amplitudes were unchanged. High-frequency SC stimulation (100 Hz for 0.5 s), or bath application of SP, resulted in gradual increases in both SC and CTX EPSP amplitudes to twofold or greater above baseline within 15–20 min poststimulation/application. This enhancement correlated with increases in input resistance and both the potentiation and resistance change were abolished in the presence of the NK1 antagonist L-703,606. These results indicate that SP is released when SC-LPN neurons fire at high frequency and SP acts postsynaptically via NK1 receptors to potentiate subsequent LPN responses to both cortical and tectal inputs. We suggest that the SP-mediated potentiation of synaptic responses may serve to amplify responses to threatening objects that move across large regions of the visual field

Distribution of synapsins and vGLUTs in the pulvinar nucleus.

<p>Tree shrew pulvinar tissue was labeled with antibodies against synapsin I or II (purple), and vGLUT1 or 2 (green). Profiles that are labeled with two antibodies appear white. Representative confocal images of the Pd (single 0.2 µm scan with a 100x objective) are illustrated. Corticopulvinar terminals (RS profiles labeled with the vGLUT1 antibody) contain synapsin I (A), but not synapsin II (C). Tectopulvinar terminals (large clusters of RM profiles labeled with the vGLUT2 antibody) contain both synapsin I (B) and synapsin II (D). Scale  =  10 µm.</p

Two types of EPSPs.

<p>A) With increasing stimulation intensity, tecto-pulvinar EPSPs in the Pd show a graded increase in amplitude. B) Average first type EPSP amplitudes and latencies as a function of stimulation intensity (n = 17), graph show a graded increase in peak amplitude correlate to the increase in stimulation current but the latency of the EPSP is not relative to stimulation current. C) Average second type EPSP amplitudes and latencies as a function of stimulation intensity (n = 8), second type EPSP show a graded increase in peak amplitude and no change in latency with increasing stimulation intensity, but the threshold amplitude was smaller and latency was longer (p<0.05).</p

Distribution of synapsins and vGLUTs in the dLGN.

<p>Tree shrew dLGN tissue was labeled with antibodies against synapsin I or II (purple), and vGLUT1 or 2 (green). Profiles that are labeled with two antibodies appear white. Representative confocal images (single 0.2 µm scan with a 100x objective) are illustrated. Corticogeniculate terminals (RS profiles labeled with the vGLUT1 antibody) contain synapsin I (A), but not synapsin II (C). Retinogeniculate terminals (RL profiles labeled with the vGLUT2 antibody) do not contain either synapsin I (B) or synapsin II (D). Scale  =  10 µm.</p

<i>In vitro</i> recording methods.

<p>A) A parasaggital section of the tree shrew brain stained for the type 2 vesicular glutamate transporter (vGLUT2) illustrates the location of the whole cell recordings in the caudal pulvinar nucleus and the location of the 8 electrode stimulus array in the stratum griseum superficial (SGS) and stratum opticum (SO) of the superior colliculus. B, C) High magnification views of the Pc (B) and Pd (C) illustrated in panel A. Immunohistochemical staining for vGLUT2 is a marker for tectopulvinar terminals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023781#pone.0023781-Chomsung2" target="_blank">[9]</a> and reveals the distinct arrangements of tectal terminals in the dorsal (Pd) central (Pc) pulvinar nucleus. The Pd contains dense clusters of tectal terminals and tubular clusters line long lengths of dendrites (C). In contrast, the Pc contains smaller more sparsely distributed clusters of tectal terminals (B). D) Voltage fluctuations recorded in response to the injection of depolarizing or hyperpolarizing current steps of varying size revealed that all recorded cells fired with both tonic action potentials, and low threshold calcium bursts. E) Drawing of a biocytin-labeled cell recorded in the juvenile pulvinar, F) Drawing of a biocytin-labeled cell recorded in the adult pulvinar. Scale in A = 1 mm. Scale in C = 30 µm and also applies to B. Scales in E and F = 10 µm. dLGN, dorsal lateral geniculate nucleus.</p