Volk, A. | bioRxiv Science

bioRxiv2023

Neural stem and progenitor cells support and protect adult hippocampal function via vascular endothelial growth factor secretion.

Adult neural stem and progenitor cells (NSPCs) reside in the dentate gyrus (DG) of the hippocampus throughout the lifespan of most mammalian species. In addition to generating new neurons, NSPCs may alter their niche via secretion of growth factors and cytokines. We recently showed that adult DG NSPCs secrete vascular endothelial growth factor (VEGF), which is critical for maintaining adult neurogenesis. Here, we asked whether NSPC-derived VEGF alters hippocampal function independent of adult neurogenesis. We found that loss of NSPC-derived VEGF acutely impaired hippocampal memory, caused neuronal hyperexcitability and exacerbated excitotoxic injury. We also found that NSPCs generate substantial proportions of total DG VEGF and VEGF disperses broadly throughout the DG, both of which help explain how this anatomically-restricted cell population could modulate function broadly. These findings suggest that NSPCs actively support and protect DG function via secreted VEGF, thereby providing a non-neurogenic functional dimension to endogenous NSPCs.

neuroscience↗

bioRxiv2021

SARS-CoV-2 Neutralization in Commercial Lots of Plasma-derived Immunoglobulin

IntroductionPatients suffering from primary or secondary immunodeficiency (PID or SID) face times of increased insecurity and discomfort in the light of the raging COVID-19 pandemic, not knowing if and to what extent their comorbidities impact the course of a potential SARS-CoV-2 infection. Furthermore, recently available vaccination options might not be amenable or effective for all patients of this heterogeneous population. Therefore, these patients often rely on passive immunization with plasma-derived, intravenous or subcutaneous immunoglobulin (IVIG/SCIG). Whether the ongoing COVID-19 pandemic and/or the progress in vaccination programs lead to increased and potentially protective titers in plasma-derived immunoglobulins (Ig) indicated, e.g., for humoral immunodeficiency remains a pressing question for this patient population. PurposeWe investigated SARS-CoV-2 reactivity of US plasma-derived IVIG/SCIG products from the end of 2020 until 06/2021 as well as in convalescent plasma (CP) from 05/2020 to 08/2020 to determine whether potentially neutralizing antibody titers may be present. MethodsFinal containers of IVIG/SCIG and CP donations were analyzed by commercial ELISA for anti-SARS-CoV-2 S1-receptor binding domain (RBD) IgG as well as microneutralization assay using a patient-derived SARS-CoV-2 (D614G) isolate. Neutralization capacities of 313 plasma single donations and 119 plasma-derived IVIG/SCIG lots were determined. Results obtained from both analytical methods were normalized against the WHO International Standard. Finally, based on dense pharmacokinetic (PK) profiles of an IVIG preparation from previously published investigations, possible steady-state plasma levels of SARS-CoV-2 neutralization capacities were approximated based on currently measured anti-SARS-CoV-2 potencies in IVIG/SCIG preparations. ResultsCP donations presented with a high variability with regards to anti-SARS-CoV-2 reactivity in ELISA as well as in neutralization testing. While approximately 50% of convalescent donations were none/low neutralizing, approximately 10% were at or above 1000 IU/mL. IVIG/SCIG lots derived from pre-pandemic plasma donations did not show neutralizing capacities of SARS-CoV-2. Lots produced between 12/2020 and 06/2021, entailing plasma donations after the emergence of SARS-CoV-2 showed a rapid and constant increase in anti-SARS-CoV-2 reactivity and neutralization capacity over time. While lot-to-lot variability was substantial, neutralization capacity increased from a mean of 20 IU/mL in 12/2020 to 505 IU/mL in 06/2021 with a maximum of 864 IU/mL for the most recent lots. Pharmacokinetic extrapolations, based on non-compartmental superposition principles using steady-state reference profiles from previously published PK investigations on IVIG in PID, yielded potential steady-state trough plasma levels of 16 IU/mL of neutralizing SARS-CoV-2 IgG based on the average final container concentration from 05/2021 of 216 IU/mL. Maximum extrapolated trough levels could reach 64 IU/mL based on the latest maximal final container potency tested in 06/2021. ConclusionsSARS-CoV-2 reactivity and neutralization capacity in IVIG/SCIG produced from US plasma rapidly and in part exponentially increased in the first half of 2021. The observed increase of final container potencies is likely trailing the serological status of the US donor population in terms of COVID-19 convalescence and vaccination by at least 5 months due to production lead times and should in principle continue at least until fall 2021. In summary, the data support rapidly increasing levels of anti-SARS-CoV-2 antibodies in IVIG/SCIG products implicating that a certain level of protection could be possible against COVID-19 for regularly substituted PID/SID patients. Nevertheless, more research is still needed to confirm which plasma levels are needed to provide protection against SARS-CoV-2 infection in immune-compromised patients. Plain Language SummaryPeople with deficiencies in their immune system often have an insufficient antibody response to antigens, e.g., bacteria, viruses, or vaccines. These patients therefore often receive antibodies from healthy people to replace the missing antibodies and build a first line of defense against infections. These antibodies (also called immunoglobulins (Ig)) are prepared from plasma of healthy donors, the liquid fraction of the blood without cells. This plasma is then split up in pharmaceutical production into its protein components. One of these is immunoglobulin G (IgG), which is the protein family that neutralizes/inactivates infectious agents as well as marks these infectious agents so they can be recognized by other parts of the immune system. With the ongoing COVID-19 pandemic and the severe to fatal outcomes for certain patient groups, especially people with impaired immunity, these patients and their physicians are interested in whether their antibody replacement therapy also confers protection against SARS-CoV-2 infection. We analyzed the capability of plasma-derived Ig lots to (i) recognize SARS-CoV-2 protein by ELISA method as well as (ii) neutralize SARS-CoV-2 by neutralization studies using the actual virus under biosafety level 3 (BSL-3) conditions. Here we show increasing anti-SARS-CoV-2 activity over time of manufactured Ig lots produced between 12/2020 and 06/2021. The most recent lots had a neutralizing activity of up to 864 IU/mL. Considering that the USA represents Octapharmas main plasma source, the progress in vaccination levels together with the evolution of the COVID-19 pandemic in this country suggests that the IVIG/SCIG neutralization capacities against SARS-CoV-2 might still increase and could potentially meet a level where antibody plasma concentrations in the patient confer immune protection. Key PointsO_LIPatients with humoral immunodeficiency rely on plasma-derived immunoglobulin for passive immunization against numerous pathogens. C_LIO_LISARS-CoV-2 neutralization capacities of plasma-derived immunoglobulins have increased over time with the ongoing COVID-19 pandemic and vaccination campaigns. C_LIO_LIPlasma-derived immunoglobulin in prophylactic use for immunodeficient patients could potentially protect against SARS-CoV-2 infection in the future. C_LI

immunology↗

bioRxiv2019

Coronavirus Interferon Antagonists Differentially Modulate the Host Response during Replication in Macrophages

Coronaviruses express a multifunctional papain-like protease, termed PLP2. PLP2 acts as a protease that cleaves the viral replicase polyprotein, and a deubiquitinating (DUB) enzyme which removes ubiquitin moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2 DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly as the wild-type virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (p<0.05) in viral titer in livers and spleens at day 5 post-infection, albeit both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of PLP2-ubiquitin complex, and that PLP2 DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis. ImportanceCoronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (de-conjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin-binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced pathogenesis in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.

microbiology↗