Aging shapes infection profiles of influenza A virus and SARS-CoV-2 in human lung slices (preprint)

The recent coronavirus disease 2019 (COVID-19) outbreak revealed the susceptibility of elderly patients to respiratory virus infections, showing cell senescence or subclinical persistent inflammatory profiles and favouring the development of severe pneumonia. In our study, we evaluated the potential influence of lung aging on the efficiency of replication of influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2), as well as determined the pro-inflammatory and antiviral responses of the distal lung tissue. Using precision-cut lung slices (PCLS) from donors of different ages, we found that pandemic H1N1 and avian H5N1 IAV replicated in the lung parenchyma with high efficacy. In contrast to these IAV strains, SARS-CoV-2 early isolate and Delta variant of concern (VOC) replicated less efficiently in PCLS. Interestingly, both viruses showed reduced replication in PCLS from older compared to younger donors, suggesting that aged lung tissue represents a sub optimal environment for viral replication. Regardless of the age-dependent viral loads, PCLS responded to infection with both viruses by an induction of IL-6 and IP-10/CXCL10 mRNAs, being highest for H5N1. Finally, while SARS-CoV-2 infection was not causing detectable cell death, IAV infection caused significant cytotoxicity and induced significant early interferon responses. In summary, our findings suggest that aged lung tissue might not favour viral dissemination, pointing to a determinant role of dysregulated immune mechanisms in the development of severe disease.

Nanoemulsions and nanocapsules as carriers for the development of intranasal mRNA vaccines (preprint)

The global emergency of coronavirus disease 2019 (COVID-19) has spurred extensive worldwide efforts to develop vaccines for protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our contribution to this global endeavor involved the development of a diverse library of nanocarriers, as alternatives to lipid nanoparticles (LNPs), including nanoemulsions (NEs) and nanocapsules (NCs), with the aim of protecting and delivering messenger ribonucleic acid (mRNA) for nasal vaccination purposes. A wide range of prototypes underwent rigorous screening through a series of in vitro and in vivo experiments, encompassing assessments of cellular transfection, cytotoxicity, and intramuscular administration of a model mRNA for protein translation. Consequently, we identified two promising candidates for nasal administration. These candidates include an NE incorporating a combination of an ionizable lipid (C12-200) and cationic lipid (DOTAP) for mRNA entrapment, along with DOPE to facilitate endosomal escape. This NE exhibited a size of 120 nm and a highly positive surface charge (+50 mV). Additionally, an NC formulation comprising the same components with a dextran sulfate shell was identified, with a size of 130 nm and a moderate negative surface charge (-16 mV). Upon intranasal administration of mRNA encoding for ovalbumin (mOVA) associated with optimized versions of NEs and NCs, robust antigen-specific CD8+ T cell responses were observed. These findings underscore the potential of NEs and polymeric NCs in advancing mRNA vaccine development for combating infectious diseases.

Discovery of anti-SARS-CoV-2 S2 protein antibody CV804 with broad-spectrum reactivity with various beta coronaviruses and analysis of its pharmacological properties in vitro and in vivo (preprint)

SARS-CoV-2 pandemic alerts us that spillovers of various animal coronaviruses to human in the future may bring us enormous damages. Thus, there is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses. CV804 against the S2 domain of the spike protein, which is less prone to mutations. CV804 shows not only broad cross-reactivities with representative 20 animal-origin coronaviruses but also with diseases-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and mutant strains of SARS-CoV-2. Other than that, the main characteristics of CV804 are that it has strong antibody-dependent cellular cytotoxicity (ADCC) activity to SARS-CoV2 spike protein-expressed cells in vitro and completely lacks virus-neutralization activity. Comprehensively in animal models, CV804 suppressed disease progression by SARS-CoV-2 infection. Structural studies using HDX-MS and point mutations of recombinant spike proteins revealed that CV804 binds to a unique epitope within the highly conserved S2 domain of the spike proteins of various coronaviruses. Based on the overall data, we suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein expressed on the surface of the infected cells and weakens the viral virulence by supporting host immune cells attack through ADCC activity in vivo. CV804 epitope identified in this study is not only useful for the design of pan-corona antibody therapeutics but also to design next-generation coronavirus vaccines and antiviral drugs.

Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome (preprint)

The COVID-19 pandemic persists despite the availability of vaccines, and it is therefore crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of the oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, while PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed the modulatory activity of oral microbial communities varied among individuals in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19.

The S2 subunit of spike encodes diverse targets for functional antibody responses to SARS-CoV-2 (preprint)

The SARS-CoV-2 virus responsible for the COVID-19 global pandemic has exhibited a striking capacity for viral evolution that drives continued evasion from vaccine and infection-induced immune responses. Mutations in the receptor binding domain of the S1 subunit of the spike glycoprotein have led to considerable escape from antibody responses, reducing the efficacy of vaccines and monoclonal antibody (mAb) therapies. Therefore, there is a need to interrogate more constrained regions of Spike, such as the S2 subdomain. Here, we describe a collection of S2 mAbs from two SARS-CoV-2 convalescent individuals that target multiple regions in the S2 subdomain and can be grouped into at least five epitope classes. Most did not neutralize SARS-CoV-2 with the exception of C20.119, which bound to a highly conserved epitope in the fusion peptide and showed broad binding and neutralization activity across SARS-CoV-2, SARS-CoV-1, and closely related zoonotic sarbecoviruses. Several of the S2 mAbs tested mediated antibody-dependent cellular cytotoxicity (ADCC) at levels similar to the S1 mAb S309 that was previously authorized for treatment of SARS-CoV-2 infections. Three of the mAbs with ADCC function also bound to spike trimers from HCoVs, such as MERS-CoV and HCoV-HKU1. Our findings suggest there are diverse epitopes in S2, including functional S2 mAbs with HCoV and sarbecovirus breadth that likely target functionally constrained regions of spike. These mAbs could be developed for potential future pandemics, while also providing insight into ideal epitopes for eliciting a broad HCoV response.

Risk of Adverse Events Following Monovalent Third or Booster Dose of COVID-19 mRNA Vaccination in U.S. Adults Ages 18 Years and Older (preprint)

Background: The U.S. FDA authorized the monovalent third primary series or booster doses of COVID-19 mRNA vaccines in August 2021 for persons 18 years and older. Monitoring of outcomes following updated authorizations is critical to evaluate vaccine safety and can provide early detection of rare adverse events (AEs) not identified in pre-licensure trials. Methods We evaluated the risk of 17 AEs following third doses of COVID-19 mRNA vaccines from August 2021 through early 2022 among adults aged 18-64 years in three commercial databases (Optum, Carelon Research, CVS Health) and adults aged >65 years in Medicare Fee-For-Service. We compared observed AE incidence rates to historical (expected) rates prior to the pandemic, estimated incidence rate ratios (IRRs) for the Medicare database and pooled IRR across the three commercial databases. Analyses were also stratified by prior history of COVID-19 diagnosis. Estimates exceeding a pre-defined threshold were considered statistical signals. Results Four AEs met the threshold for statistical signals for BNT162b2 and mRNA-1273 vaccines including Bells Palsy and pulmonary embolism in Medicare, and anaphylaxis and myocarditis/pericarditis in commercial databases. Nine AEs and three AEs signaled among adults with and without prior COVID-19 diagnosis, respectively. Conclusions This early monitoring study identified statistical signals for AEs following third doses of COVID-19 mRNA vaccination. Since this method is intended for screening purposes and generates crude results, results do not establish a causal association between the vaccines and AEs. FDAs public health assessment remains consistent that the benefits of COVID-19 vaccination outweigh the risks of vaccination.

Mathematical Modelling Indicates Th-cell Targeted Antibody-Dependent Cellular Cytotoxic Is a Crucial Obstacle Hurdling HIV Vaccine Development (preprint)

HIV poses a significant threat to human health. Although some progress has been made in the development of an HIV vaccine, there is currently no reported success in achieving an effective and fully functional vaccine for HIV. This highlights the challenges involved in HIV vaccine development. Through mathematical modeling, we have conducted a systematic study on the impact of antibody-dependent cellular cytotoxicity (ADCC) on HIV-specific immune responses. Unlike other viral infections, the ADCC effect following HIV infection may cause significant damage to the follicular center Th cells, leading to apoptosis of follicular center cells and rapid death of effector Th cells. This impedes the generation of neutralizing antibodies and creates barriers to viral clearance, thereby contributing to long-term infection. Another challenge posed by this effect is the substantial reduction in vaccine effectiveness, as effective antigenic substances such as gp120 bind to Th cell surfaces, resulting in the apoptosis of follicular center Th cells due to ADCC, hindering antibody regeneration. To address this issue, we propose the concept of using bispecific antibodies. By genetically editing B cells to insert the bispecific antibody gene, which consists of two parts targeting the CD4 binding site of HIV, such as the broadly neutralizing antibody 3BNC117, and the other targeting antibodies against other viruses, such as the spike protein of SARS-CoV-2. We can simultaneously enhance the levels of two pathogen-specific antibodies through stimulation with non-HIV-antigens corresponding to the other part of the chimeric antibody, such as the spike protein. This study contributes to the elucidation of the pathophysiology of HIV, while also providing a theoretical framework for the successful development of an HIV vaccine.

Hydroxychloroquine safety, comparison between no Covid-19 and Covid-19 patients. Data from the Spanish Pharmacovigilance Database (preprint)

Aim: To compare the cases reported to the Spanish Pharmacovigilance System (SEFV-H) with HCQ used in COVID-19 vs. HCQ used in other indications. Methods. All cases of adverse drug reactions (ADR) submitted to the Spanish Pharmacovigilance database (FEDRA) from 1 January 1982 to 19 February 2021 suspected to be induced by HCQ were identified. Cases were classified into two groups: no-Covid patients and Covid patients. Frequencies of ADR were compared. Reporting Odds Ratios (ROR) with its lower limit of the 95% confidence interval (-ROR) and Omega (Ω) and its lower limit of the 95% credibility interval (Ω -025) were obtained to estimate disproportionalities. Results. More severe cases were reported with the use of HCQ in Covid. Main differences in frequency were observed in hepatobiliary, skin, gastrointestinal, eye, nervous system and heart ADRs. During the Covid-19 pandemic, disproportionality was found for Torsade de Pointes/QT prolongation with a ROR (-ROR) of 132.8 (76.7); severe hepatotoxicity, 18.7 (14.7); dyslipidaemias, 12.1 (6.1); shock, 9.5 (6.9) and ischaemic colitis, 8.9 (2.6). Myopathies, haemolytic disorders and suicidal behaviour increased their disproportionality during the pandemic. Disproportionality was observed for neoplasms, haematopoietic cytopaenias and interstitial lung disease in the pre-Covid period. Ω showed potential interactions between HCQ and azithromycin, ceftriaxone, lopinavir and tocilizumab . Conclusions. The use of HCQ in Covid-19 changed its safety profile. Of particular concern during the pandemic were arrhythmias, hepatotoxicity, severe skin reactions and suicide risk, but not ocular disorders. Some ADRs identified as signals would require more detailed analyses.

Allergic reactions to the Ad26.COV2.S Vaccine in South Africa (preprint)

Abstract ( n=254/250 words)   Background: The Janssen-Ad26.COV2.S vaccine is authorised for use in several countries with more than 30 million doses administered. Mild and severe allergic adverse events following immunisation(AEFI) have been reported. The aim of this report is to detail allergic reactions reported during the Sisonke phase 3B study in South Africa. Methods: : A single-dose of the Ad26.COV2.S vaccine was administered to 477234 South African Healthcare Workers between 17 February and 17 May 2021. Monitoring of adverse events used a combination of passive reporting and active case finding. Telephonic contact was attempted for all adverse events reported as “allergy”. Anaphylaxis adjudication was performed using the Brighton Collaboration (BCC) and NIAID case definitions.  Results: : A large cohort of South African healthcare workers received the Ad26.COV2.S vaccination. Only 250(0.052%) patients reported any allergic-type reaction(less than 1 in 2000), with four cases of adjudicated anaphylaxis (BCC level 1, n=3)(prevalence of 8.4 per million doses). All anaphylaxis cases had a prior history of drug or vaccine-associated anaphylaxis. Cutaneous allergic reactions were the commonest non-anaphylatic reactions and included: self-limiting, transient/localised rashes requiring no healthcare contact(n=91); or isolated urticaria and/or angioedema[n=70 median  onset 48(IQR 11.5-120) hours post vaccination] that necessitated healthcare contact(81%), antihistamine(63%), and/or systemic/topical corticosteroid(16%). All immediate (including adjudicated anaphylaxis) and the majority of delayed AEFI(65/69) cases resolved completely.   Conclusions: : Allergic AEFI are rare following a single-dose of Ad26.COV with complete resolution in  all cases of anaphylaxis. Though rare, isolated, delayed onset urticaria and/or angioedema was the commonest allergic AEFI requiring treatment, with nearly half occurring in participants without known atopic disease.   Keywords: allergic reaction, anaphylaxis, COVID19 adenovirus vaccine; Janssen-Ad26.COV2.S vaccine, urticaria

Epitopic mining on Spike protein of SARS-CoV-2 as a candidate target for vaccine design: An in-silico analysis (preprint)

The outbreak of novel SARS-CoV-2 virion has wreaked havoc with a high prevalence of respiratory illness and high transmission due to a vague understanding of the viral antigenicity augmenting dire challenge to public health globally. This viral member requisite the expansion of diagnostic and therapeutic tools to track its transmission and confront through vaccine development. Therefore, prophylactic strategies are mandatory. Virulence-related spike proteins can be the desirable candidate befitting computational design of vaccines targeting SARS-CoV-2 followed by meteoric development of immune epitopes. This study aims to characterize Spike protein using the existing knowledge related to the immunological profile of SARS-CoV-2 to predict immunogenic epitopes based on antigenicity, allergenicity, toxicity, immunogenicity, and population coverage. Applying in-silico approaches, a set of twenty-four B lymphocyte-based epitopes and forty-six T lymphocyte-based epitopes (MHC-I and MHC-II) were selected. The predicted epitopes were evaluated for their intrinsic properties. Physico-chemical characterization of epitopes qualify them for further in vitro and in vivo analysis pre-requisite vaccine development. This study presents a set of screened epitopes that binds to the HLA- specific allelic proteins that can be employed for designing a multi-epitopic peptide vaccine construct (MEPVC) against SARS-CoV-2 that will confer vaccine-induced protective immunity due to its structural stability.

Transcriptome Heterogeneity in COVID-19-induced Acute Respiratory Distress Syndrome (preprint)

COVID-19 is a major etiology of acute respiratory distress syndrome (ARDS). The biological phenotypes and underlying mechanisms in COVID-19-induced ARDS are not fully understood. Bronchoalveolar lavage fluid (BALF) cells and clinical data were collected from patients with COVID-19-induced ARDS. Principal component analysis of genome-wide expression data obtained from bulk RNA sequencing of BALF cells subgrouped COVID-19-induced ARDS patients. Moreover, comparing transcriptome profiles between the subgroups showed two biological phenotypes, illustrated by up- and down-regulation of interferon (IFN) responses, despite no significant differences in clinical characteristics including onset and outcomes. In the low-IFN phenotype, in contrast to the high-IFN phenotype, the TLR-MyD88-IFN regulatory factor (IRF) 5 and cGAS-STING1 axes related to type Ⅰ IFN and the IRF8-interleukin (IL)-12-STAT4 and IRF1-IL-15-DNAX-activation protein 10 axes related to type Ⅱ IFN were inactivated at the transcriptional level, together with the PERK-C/EBP homologous protein axis and the IL-10-hemoglobin scavenger receptor CD163 axis. The pathogenesis of ARDS in the low-IFN phenotype was illustrated by damage to type II alveolar epithelial cells due to increased viral replication by reduced antiviral response, cytotoxicity, and apoptotic signaling and impaired free hemoglobin catabolism. Our data uncovered heterogeneous IFN responses, the underlying mechanisms, and related pathogenesis in COVID-19-induced ARDS.

Extended Duration and Intense Manifestations of COVID-19-Linked Cytotoxic Lesions of the Corpus Callosum: Case Reports and Literature Review (preprint)

While clinical instances of cytotoxic lesions of the corpus callosum (CLOCCs) are well-documented, international reports specific to COVID-19-related cases remain limited. This paper presents the case of a 40-year-old female patient admitted due to "sudden dizziness and poor limb coordination for 7 weeks following fever." She tested positive for COVID-19 and experienced symptoms like dizziness, temporary confusion, nausea, vomiting, cerebellar speech issues, and ataxia after fever onset. Later, she developed pyramidal tract symptoms and behavioral abnormalities. Head MRI revealed abnormal high signal in the splenium of the corpus callosum and abnormal signals in the left cerebellar peduncle on DWI. With no significant medical history and exclusion of other causes during treatment (including steroid therapy and two doses of intravenous immunoglobulin), a follow-up MRI after one month showed the lesions had disappeared. However, clinical recovery was slow, with residual symptoms persisting for almost a year, including involuntary tremors in the upper limbs and head. Phenytoin, gabapentin, and pregabalin showed limited effectiveness in treatment, but Arotinolol and donepezil led to slight improvement in involuntary tremors. This case suggests that COVID-19-associated CLOCCs might have a protracted course and severe symptoms, demanding differentiation from ischemic cerebrovascular diseases, particularly in early stages.

Altered IgG4 Antibody Response to Repeated mRNA versus Protein COVID Vaccines (preprint)

Repeated mRNA SARS-CoV-2 vaccination has been associated with increases in the proportion of IgG4 in spike-specific antibody responses and concurrent reductions in Fc{gamma}-mediated effector functions that may limit control of viral infection. Here, we assessed anti-Spike total IgG, IgG1, IgG2, IgG3 and IgG4, and surrogate markers for antibody-dependent cellular phagocytosis (ADCP, Fc{gamma}RIIa binding), antibody-dependent cellular cytotoxicity (ADCC, Fc{gamma}RIIIa binding), and antibody-dependent complement deposition (ADCD, C1q binding) associated with repeated SARS-CoV-2 vaccination with NVX-CoV2373 (Novavax Inc., Gaithersburg, MD). The NVX-CoV2373 protein vaccine did not induce notable increases in spike-specific IgG4 or negatively impact surrogates for Fc{gamma} effector responses. Conversely, repeated NVX-CoV2373 vaccination uniquely enhanced IgG3 responses which are known to exhibit strong affinity for Fc{gamma}RIIIa and have previously been linked to potent neutralization of SARS-CoV-2. Subsequent investigations will help to understand the immunological diversity generated by different SARS-CoV-2 vaccine types and have the potential to reshape public health strategies.

Comprehensive procedure for injecting Evusheld® for hematological diseases in a single institute (preprint)

Tixagevimab and cilgavimab (EVA, Evusheld®), monoclonal antibody combination treatments, consisted of two neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). EVA showed prophylactic and therapeutic effects against coronavirus disease 2019. The Japanese Society of Hematology recommended EVA for such patients with active treatment, but each institution decided on comprehensive administration. We develop a systematic procedure for comprehensive EVA injection in patients with hematological malignancies without any over/under-indication. We listed all patients with the required indications from November 2022 to March 2023. We included 178 cases, 84 females and 94 males, with a median age of 70 (range: 19–90) years. Underlying diseases are myeloid neoplasms in 36 (20%), lymphoid neoplasms in 75 (73%), and others. Indications were intensively hematological malignancy treatment, rituximab treatment within 12 months, burton kinase inhibitor treatment, after chimeric antigen receptor T cell immunotherapy, and after stem cell transplantation in 74 (41%), 73 (41%), 3 (2%), 5 (3%), and 23 (13%) cases, respectively. Of the 178 cases, 22 (12.4%) refused EVA injection. Further, 42 and 136 cases were administered outpatient and inpatient, respectively. Over 95% of the listed cases received EVA injection within 3 months. No severe toxicities were observed among them (N = 156), and 8 (5.2%) cases had breakthrough SARS-CoV-2 infection, which was significantly lower (P = 0.02) than those without EVA (4 [18.2%] of 22 cases). Both groups showed no moderate or severe infection cases. This single-center experience showed that comprehensive EVA injection management effectively generated safer completion with preferable clinical impact.

Assessing heating efficiencies of PVPylated divalent metal-doped MFe2O4 nanoparticles for magnetic hyperthermia (preprint)

There is an incessant demand to keep improving on the heating responses of polymeric magnetic nanoparticles (MNPs) under magnetic excitation, particularly in their pursuit to be utilized for clinical hyperthermia applications. Herein, we report the fabrication of a panel of PVP-coated metal-doped MFe2O4 (M ≅ Co, Ni, Mn, Zn) MNPs prepared via the Ko-precipitation Hydrolytic Basic (KHB) methodology and assess their magnetic and self-heating abilities. The physiochemical, structural, morphological, compositional, and magnetic properties of the doped MNPs were fully characterized using various spectroscopic techniques mainly TEM, XRD, FTIR, and VSM. The obtained MNPs exhibited stabilized quasi-spherical sized particles (10–15 nm), well-crystallized cubic inverse spinel phases, high saturation magnetizations (26–81 emu/g) and ferromagnetic behavior. In response to alternating magnetic field (AMF), distinctive heating responses of these doped ferrite NPs were attained. Heating efficacies and specific absorption rate (SAR) values as functions of concentration, frequency, and amplitude were systematically investigated. The highest heating performance was observed for PVP-NiFe2O4 followed by PVP-CoFe2O4 and the least for PVP-Zn-doped and Mn-doped MNPs (SAR values Ni > Co > Zn > Mn). Finally, cytotoxicity assay was conducted on aqueous dispersions of the doped ferrite NPs, proving their biocompatibility and low toxicity. Our results strongly suggest that the PVPylated metal-doped ferrite NPs prepared here, particularly Ni- and Co-doped MNPs, are promising vehicles for potential combined magnetically-triggered biomedical hyperthermia applications.

mRNA-LNP COVID-19 vaccine lipids induce low level complement activation and production of proinflammatory cytokines: Mechanisms, effects of complement inhibitors, and relevance to adverse reactions (preprint)

Messenger RNA-containing lipid nanoparticles (mRNA-LNPs) enabled widespread COVID-19 vaccination with a small fraction of vaccine recipients displaying acute or sub-acute inflammatory symptoms. The molecular mechanism of these adverse events (AEs) remains undetermined. Here we report that the mRNA-LNP vaccine, Comirnaty, triggers low-level complement (C) activation and production of inflammatory cytokines, which may be key underlying processes of inflammatory AEs. In serum, Comirnaty and the control PEGylated liposome (Doxebo) caused different rises of C split products, C5a, sC5b-9, Bb and C4d, indicating stimulation of the classical pathway of C activation mainly by the liposomes, while a stronger stimulation of the alternative pathway was equal with the vaccine and the liposomes. Spikevax had similar C activation as Comirnaty, but viral or synthetic mRNAs had no such effect. In autologous serum-supplemented peripheral blood mononuclear cell (PBMC) cultures, Comirnaty caused increases in the levels of sC5b-9 and proinflammatory cytokines in the following order: IL-1 < IFN-{gamma} < IL-1{beta} < TNF- < IL-6 < IL-8, whereas heatinactivation of serum prevented the rises of IL-1, IL-1{beta}, and TNF-. Clinical C inhibitors, Soliris and Berinert, suppressed vaccine-induced C activation in serum but did not affect cytokine production when applied individually. These findings suggest that the PEGylated lipid coating of mRNA-LNP nanoparticles can trigger C activation mainly via the alternative pathway, which may be causally related to the induction of some, but not all inflammatory cytokines. While innate immune stimulation is essential for the vaccines efficacy, concurrent production of C- and PBMC-derived inflammatory mediators may contribute to some of the AEs. Pharmacological attenuation of harmful cytokine production using C inhibitors likely requires blocking the C cascade at multiple points.

Longitudinal transcriptional changes reveal genes from the natural killer cell-mediated cytotoxicity pathway as critical players underlying COVID-19 progression (preprint)

Patients present a wide range of clinical severities in response SARS-CoV-2 infection, but the underlying molecular and cellular reasons why clinical outcomes vary so greatly within the population remains unknown. Here, we report that negative clinical outcomes in severely ill patients were associated with divergent RNA transcriptome profiles in peripheral immune cells compared with mild cases during the first weeks after disease onset. Protein-protein interaction analysis indicated that early-responding cytotoxic NK cells were associated with an effective clearance of the virus and a less severe outcome. This innate immune response was associated with the activation of select cytokine-cytokine receptor pathways and robust Th1/Th2 cell differentiation profiles. In contrast, severely ill patients exhibited a dysregulation between innate and adaptive responses affiliated with divergent Th1/Th2 profiles and negative outcomes. This knowledge forms the basis of clinical triage that may be used to preemptively detect high-risk patients before life-threatening outcomes ensue. Highlights- Mild COVID-19 patients presented an early compromise with NK cell function, whereas severe patients do so with neutrophil function. - The identified co-expressed genes give insights into a coordinated transcriptional program of NK cell cytotoxic activity being associated with mild patients. - Key checkpoints of NK cell cytotoxicity that were enriched in mild patients include: KLRD1, CD247, and IFNG. - The early innate immune response related to NK cells connects with the Th1/Th2 adaptive immune responses, supporting their relevance in COVID-19 progression.

Cold Atmospheric Helium Plasma In The Post-Covid Era: A Promising Tool For The Disinfection Of Silicone Endotracheal Prostheses (preprint)

The COVID-19 pandemic resulted in a high prevalence of laryngotracheal stenosis. The endoluminal tracheal prostheses used to treat this condition are made of medical-grade silicone (MGS). Despite their excellent properties, the main limitation of these prostheses is the formation of a polymicrobial biofilm on their surfaces that interacts with the underlying mucosa, causing local inflammation and interfering with the local healing process, ultimately leading to further complications in the clinical scenario. Cold atmospheric plasma (CAP) shows antibiofilm properties on several microbial species. The present study evaluated the inhibitory effect of CAP on multispecies biofilms grown on MGS surfaces. In addition to the MGS characterization before and after CAP exposure, the cytotoxicity of CAP on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the MGS surface wetting characteristics from hydrophilic (80.5 degrees) to highly hydrophilic (< 5 degrees). ATR-FTIR shows no significant alterations in the surficial chemical composition of MGS before and after CAP exposure for 5 min. A significant log reduction of viable cells in mono-species biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) were detected after CAP exposure. Multi-species biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. It could be concluded that CAP can be a simple and effective method to delay the multi-species biofilm formation inside the endotracheal prosthesis.

Discovery of anthelmintic small molecules in the Medicines for Malaria Ventures COVID and Global Health Priority Boxes using an infrared-based assay for Caenorhabditis elegans motility (preprint)

Background: Parasitic nematodes are a public health problem globally, and an economic burden on animal and plant agricultural industries. With their ability to generate drug resistance, new anthelmintic compounds must be constantly sourced. Methods: Using the free-living nematode, Caenorhabditis elegans, in an infrared-based motility assay, we screened 400 compounds from two open-source, small-molecule collections distributed by the Medicines for Malaria Venture, namely, the COVID Box and Global Health Priority Box. The screening assay was first validated for worm number, DMSO concentration and final volume. Results: Primary and secondary (time- and concentration-dependent) screens of both boxes, identified twelve compounds as hits; nine of which were known anthelmintics. Three novel anthelmintic hits, flufenerim, flucofuron and indomethacin were identified with EC50 values ranging from 0.211 to 23.174 M. Counter toxicity screens with HEK293 cells indicated varying degrees of toxicity with EC50 values ranging from 0.453 to >100 M. Conclusions: A C. elegans motility assay was optimized and used to screen two recently-released, small molecule libraries. One or more of these three novel active compounds might serve as starting points for the development of new anthelmintics.

Overall Health Effects of mRNA COVID-19 Vaccines in Children and Adolescents: A Systematic Review and Meta-Analysis (preprint)

Importance: Phase 3 randomized controlled trials (RCTs) of mRNA COVID-19 vaccines in children and adolescents showed efficacy in preventing COVID-19 infections. Vaccines may have non-specific effects; this is not assessed within the current regulatory framework. Objective: Conduct a systematic review and meta-analysis of the phase 3 trials to assess overall and non-specific health effects of the mRNA COVID-19 vaccines in children. Data Sources: PubMed, Embase, Clinical Trials, Web of Science, and regulatory websites were searched for RCTs of mRNA vaccines. The latest trial data was included. Study Selection: All RCTs conducted with mRNA vaccines BNT162b2 and mRNA-1237 in children and adolescents below 18 years of age, with placebo, adjuvant, or other vaccines as controls. Two authors independently screened 1199 studies, six were included in the analysis. Data Extraction and Synthesis: Data on serious adverse events (SAEs) and severe adverse events (AEs) as well as organ-specific diseases was extracted following the PRISMA reporting guideline, with a focus on non-specific infectious events. Risk Ratios (RRs) comparing vaccine and placebo cohorts were calculated separately for each vaccine and combined in Mantel-Haenszel estimates. Main Outcomes and Measures: The primary outcome was SAEs, overall and infectious. Secondary outcomes were severe AEs and lower respiratory tract infection (LRTI) including RSV. Results: The analyses included 25,549 individuals (17,538 mRNA vaccine recipients and 8,011 placebo recipients). The risk of SAEs was similar for vaccine and placebo recipients. Both mRNA vaccines were associated with increased risk of severe AEs in older children. In a combined analysis, the RR was 3.77 (1.56-9.13[0.4% vs 0.1% in vaccine vs placebo recipients]) in above 5 year-olds, and 0.82 (0.53-1.29)[0.8% vs 0.9%])in younger children (p=0.003 for same effect in older and younger children). In the younger children, mRNA vaccines were associated with higher risk of LRTI (RR=3.03 (1.29-7.09)[0.6% vs 0.2%]). Conclusions and Relevance: mRNA vaccines did not increase the risk of SAEs but were associated with an increased risk of severe AEs in older children, and an increased risk of LRTI in the young. Further research into the overall and non-specific health effects of mRNA vaccines is warranted.