Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine (preprint)

ABSTRACT Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle–immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines, or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0 to 70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68 to 16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9 to 377.1) and 2.64-fold (0.76 to 12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo . PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA-vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle–leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA-vaccines should be monitored.

Integrated immune networks in SARS-CoV-2 infected pregnant women reveal differential NK cell and unconventional T cell activation (preprint)

Although pregnancy poses a greater risk for severe COVID-19, the underlying immunological changes associated with SARS-CoV-2 infection during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in pregnant and non-pregnant women during acute and convalescent COVID-19 up to 258 days post symptom onset, quantifying 217 immunological parameters. Additionally, matched maternal and cord blood were collected from COVID-19 convalescent pregnancies. Although serological responses to SARS-CoV-2 were similar in pregnant and non-pregnant women, cellular immune analyses revealed marked differences in key NK cell and unconventional T cell responses during COVID-19 in pregnant women. While NK, γδ T cells and MAIT cells displayed pre-activated phenotypes in healthy pregnant women when compared to non-pregnant age-matched women, activation profiles of these pre-activated NK and unconventional T cells remained unchanged at acute and convalescent COVID-19 in pregnancy. Conversely, activation dynamics of NK and unconventional T cells were prototypical in non-pregnant women in COVID-19. In contrast, activation of αβ CD4 + and CD8 + T cells, T follicular helper cells and antibody-secreting cells was similar in pregnant and non-pregnant women with COVID-19. Elevated levels of IL-1β, IFN-γ, IL-8, IL-18 and IL-33 were also found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, our study provides the first comprehensive map of longitudinal immunological responses to SARS-CoV-2 infection in pregnant women, providing insights into patient management and education during COVID-19 pregnancy.

Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity (preprint)

Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleural fluid) from hospitalized COVID-19 patients and non-COVID-19 controls. Cellular, humoral and cytokine responses were analysed and correlated with clinical data. SARS-CoV-2-specific IgM, IgG and IgA antibodies were detected using ELISA and multiplex assay in both the respiratory tract and blood of COVID-19 patients, although a higher receptor binding domain (RBD)-specific IgM and IgG seroconversion level was found in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples was detected only when high levels of RBD-specific antibodies were present. Strikingly, cytokine/chemokine levels and profiles greatly differed between respiratory samples and plasma, indicating that inflammation needs to be assessed in respiratory specimens for the accurate assessment of SARS-CoV-2 immunopathology. Diverse immune cell subsets were detected in respiratory samples, albeit dominated by neutrophils. Importantly, we also showed that dexamethasone and/or remdesivir treatment did not affect humoral responses in blood of COVID-19 patients. Overall, our study unveils stark differences in innate and adaptive immune responses between respiratory samples and blood and provides important insights into effect of drug therapy on immune responses in COVID-19 patients.

Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity (preprint)

Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleural fluid) from hospitalized COVID-19 patients and non-COVID-19 controls. Cellular, humoral and cytokine responses were analysed and correlated with clinical data. SARS-CoV-2-specific IgM, IgG and IgA antibodies were detected using ELISA and multiplex assay in both the respiratory tract and blood of COVID-19 patients, although a higher receptor binding domain (RBD)-specific IgM and IgG seroconversion level was found in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples was detected only when high levels of RBD-specific antibodies were present. Strikingly, cytokine/chemokine levels and profiles greatly differed between respiratory samples and plasma, indicating that inflammation needs to be assessed in respiratory specimens for the accurate assessment of SARS-CoV-2 immunopathology. Diverse immune cell subsets were detected in respiratory samples, albeit dominated by neutrophils. Importantly, we also showed that dexamethasone and/or remdesivir treatment did not affect humoral responses in blood of COVID-19 patients. Overall, our study unveils stark differences in innate and adaptive immune responses between respiratory samples and blood and provides important insights into effect of drug therapy on immune responses in COVID-19 patients.

Integrated immune networks in SARS-CoV-2 infected pregnant women reveal differential NK cell and unconventional T cell activation (preprint)

Although pregnancy poses a greater risk for severe COVID-19, the underlying immunological changes associated with SARS-CoV-2 infection during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in pregnant and non-pregnant women during acute and convalescent COVID-19 up to 258 days post symptom onset, quantifying 217 immunological parameters. Additionally, matched maternal and cord blood were collected from COVID-19 convalescent pregnancies. Although serological responses to SARS-CoV-2 were similar in pregnant and non-pregnant women, cellular immune analyses revealed marked differences in key NK cell and unconventional T cell responses during COVID-19 in pregnant women. While NK cells, {gamma}{delta} T cells and MAIT cells displayed pre-activated phenotypes in healthy pregnant women when compared to non-pregnant age-matched women, activation profiles of these pre-activated NK and unconventional T cells remained unchanged at acute and convalescent COVID-19 in pregnancy. Conversely, activation dynamics of NK and unconventional T cells were prototypical in non-pregnant women in COVID-19. In contrast, activation of {beta} CD4+ and CD8+ T cells, T follicular helper cells and antibody-secreting cells was similar in pregnant and non-pregnant women with COVID-19. Elevated levels of IL-1{beta}, IFN-{gamma}, IL-8, IL-18 and IL-33 were also found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, our study provides the first comprehensive map of longitudinal immunological responses to SARS-CoV-2 infection in pregnant women, providing insights into patient management and education during COVID-19 pregnancy.

A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2 (preprint)

As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus; whether through infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure SARS-CoV-2 immunity, ideally with rapid turnaround and without the need for laboratory-based testing. Current rapid point-of-care (POC) tests measure antibodies (Ab) against the SARS-CoV-2 virus, however, these tests provide no information on whether the antibodies can neutralise virus infectivity and are potentially protective, especially against newly emerging variants of the virus. Neutralising Antibodies (NAb) are emerging as a strong correlate of protection, but most current NAb assays require many hours or days, samples of venous blood, and access to laboratory facilities, which is especially problematic in resource-limited settings. We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibodies from whole blood, with a result that can be determined by eye (semi-quantitative) or on a small instrument (quantitative), and results show high correlation with microneutralisation assays. This assay also provides a measure of total anti-RBD antibody, thereby providing evidence of exposure to SARS-CoV-2 or immunisation, regardless of whether NAb are present in the sample. By testing samples from immunised macaques, we demonstrate that this test is equally applicable for use with animal samples, and we show that this assay is readily adaptable to test for immunity to newly emerging SARS-CoV-2 variants. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (R 2 =0.75, p<0.0001), and that fingerprick whole blood samples are sufficient for this test. Accordingly, the COVID-19 NAb-test™ device described here can provide a rapid readout of immunity to SARS-CoV-2 at the point of care.

Altered microRNA expression in COVID-19 patients enables identification of SARS-CoV-2 infection (preprint)

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management

Single-cell RNA Expression of SARS-CoV-2 Cell Entry Factors in Human Endometrium during Preconception (preprint)

We investigated potential SARS-CoV-2 tropism in human endometrium by single-cell RNA-sequencing of viral entry-associated genes in healthy women. Percentages of endometrial cells expressing ACE2, TMPRSS2, CTSB, or CTSL were <2%, 12%, 80%, and 80%, respectively, with 0.7% of cells expressing all four genes. Our findings imply low efficiency of SARS-CoV-2 infection in the endometrium before embryo implantation, providing information to assess preconception risk in asymptomatic carriers.

Ambroxol Hydrochloride Inhibits the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein's Receptor Binding Domain and Recombinant Human ACE2. (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), enters the host cells through two main pathways, both involving key interactions between viral envelope-anchored spike glycoprotein of the novel coronavirus and the host receptor, angiotensin-converting enzyme 2 (ACE2). To date, SARS-CoV-2 has infected up to 26 million people worldwide; yet, there is no clinically approved drug or vaccine available. Therefore, a rapid and coordinated effort to re-purpose clinically approved drugs that prevent or disrupt these critical entry pathways of SARS-CoV-2 spike glycoprotein interaction with human ACE2, could potentially accelerate the identification and clinical advancement of prophylactic and/or treatment options against COVID-19, thus providing possible countermeasures against viral entry, pathogenesis and survival. Herein, we discovered that Ambroxol hydrochloride (AMB), and its progenitor, Bromhexine hydrochloride (BHH), both clinically approved drugs are potent effective modulators of the key interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and human ACE2. We also found that both compounds inhibited SARS-CoV-2 infection-induced cytopathic effect at micromolar concentrations. Therefore, in addition to the known TMPRSS2 activity of BHH; we report for the first time that the BHH and AMB pharmacophore has the capacity to target and modulate yet another key protein-protein interaction essential for the two known SARS-CoV-2 entry pathways into host cells. Altogether, the potent efficacy, excellent safety and pharmacologic profile of both drugs along with their affordability and availability, makes them promising candidates for drug repurposing as possible prophylactic and/or treatment options against SARS-CoV-2 infection.

Non-permissive SARS-CoV-2 infection of neural cells in the developing human brain and neurospheres (preprint)

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that many other biological systems are affected, including the central nervous system (CNS). Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but its neurotropic potential is still debated. Here, we investigate the presence of SARS-CoV-2 in the brain from an infant patient deceased from COVID-19. The susceptibility to virus infection was compatible with the expression levels of viral receptor ACE2, which is increased in the ChP in comparison to other brain areas. To better comprehend the dynamics of the viral infection in neural cells, we exposed human neurospheres to SARS-CoV-2. Similarly to the human tissue, we found viral RNA in neurospheres, although viral particles in the culture supernatant were not infective. Based on our observations in vivo and in vitro, we hypothesize that SARS-CoV-2 does not generate productive infection in developing neural cells and that infection of ChP weakens the blood-cerebrospinal fluid barrier allowing viruses, immune cells, and cytokines to access the CNS, causing neural damage in the young brain.

SARS CoV-2 nucleocapsid protein forms condensates with viral genomic RNA (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with unstructured RNA, it forms mesh like-structures with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified an intrinsically-disordered region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. By screening 1,200 FDA approved drugs in vitro, we identified a kinase inhibitor nilotinib, which affects the morphology of N condensates in vitro and disrupts phase separation of the N protein in vivo. These results indicate that the N protein compartmentalizes viral RNA in infected cells through liquid-liquid phase separation, and this process can be disrupted by a possible drug candidate.

A dual antigen ELISA allows the assessment of SARS-CoV-2 antibody seroprevalence in a low transmission setting (preprint)

Estimates of seroprevalence of SARS-CoV-2 antibodies have been hampered by inadequate assay sensitivity and specificity. Using an ELISA-based approach to that combines data about IgG responses to both the Nucleocapsid and Spike-receptor binding domain antigens, we show that near-optimal sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (0 to 0.72%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells (preprint)

Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle. One-sentence summarySARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.

Identification of potential key genes for SARS-CoV-2 infected human bronchial organoids based on bioinformatics analysis (preprint)

There is an urgent need to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) that leads to COVID-19 and respiratory failure. Our study is to discover differentially expressed genes (DEGs) and biological signaling pathways by using a bioinformatics approach to elucidate their potential pathogenesis. The gene expression profiles of the GSE150819 datasets were originally produced using an Illumina NextSeq 500 (Homo sapiens). KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) were utilized to identify functional categories and significant pathways. KEGG and GO results suggested that the Cytokine-cytokine receptor interaction, P53 signaling pathway, and Apoptosis are the main signaling pathways in SARS-CoV-2 infected human bronchial organoids (hBOs). Furthermore, NFKBIA, C3, and CCL20 may be key genes in SARS-CoV-2 infected hBOs. Therefore, our study provides further insights into the therapy of COVID-19.

Suboptimal SARS-CoV-2-specific CD8+ T-cell response associated with the prominent HLA-A*02:01 phenotype (preprint)

An improved understanding of human T-cell-mediated immunity in COVID-19 is important if we are to optimize therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T-cell memory to shared peptides presented by common HLA types like HLA-A2. Following re-infection, cross-reactive CD8+ T-cells enhance recovery and diminish clinical severity. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from SARS-CoV-2 Spike, Nucleocapsid and Membrane proteins led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T-cells in vitro, with CD4+ sets being typically robust. For CD8+ T-cells taken directly ex vivo, we identified two HLA-A*02:01-restricted SARS-CoV-2 epitopes, A2/S269-277 and A2/Orf1ab3183-3191. Using peptide-HLA tetramer enrichment, direct ex vivo assessment of the A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that the more prominent A2/S269+CD8+ set was detected at comparable frequency ([~]1.3x10-5) in acute and convalescent HLA-A*02:01+ patients. But, while the numbers were higher than those found in uninfected HLA-A*02:01+ donors ([~]2.5x10-6), they were low when compared with frequencies for influenza-specific (A2/M158) and EBV-specific (A2/BMLF1280) ([~]1.38x10-4) populations. Phenotypic analysis ex vivo of A2/S269+CD8+ T-cells from COVID-19 convalescents showed that A2/S269+CD8+ T-cells were predominantly negative for the CD38, HLA-DR, PD-1 and CD71 activation markers, although the majority of total CD8+ T-cells were granzyme and/or perforin-positive. Furthermore, the bias towards naive, stem cell memory and central memory A2/S269+CD8+ T-cells rather than effector memory populations suggests that SARS-CoV2 infection may be compromising CD8+ T-cell activation. Priming with an appropriate vaccine may thus have great value for optimizing protective CD8+ T-cell immunity in COVID-19.

NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge (preprint)

There is an urgent need for a safe and protective vaccine to control the global spread of SARS-CoV-2 and prevent COVID-19. Here, we report the immunogenicity and protective efficacy of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length SARS-CoV-2 spike (S) glycoprotein stabilized in the prefusion conformation. Cynomolgus macaques (Macaca fascicularis) immunized with NVX-CoV2373 and the saponin-based Matrix-M adjuvant induced anti-S antibody that was neutralizing and blocked binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. Following intranasal and intratracheal challenge with SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support ongoing phase 1/2 clinical studies of the safety and immunogenicity of NVX-CoV2327 vaccine (NCT04368988). HighlightsO_LIFull-length SARS-CoV-2 prefusion spike with Matrix-M1 (NVX-CoV2373) vaccine. C_LIO_LIInduced hACE2 receptor blocking and neutralizing antibodies in macaques. C_LIO_LIVaccine protected against SARS-CoV-2 replication in the nose and lungs. C_LIO_LIAbsence of pulmonary pathology in NVX-CoV2373 vaccinated macaques. C_LI