scholarly journals Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

2021 ◽  
Author(s):  
Juanjie Tang ◽  
Your Lee ◽  
Supriya Ravichandran ◽  
Gabrielle Grubbs ◽  
Chang Huang ◽  
...  
Keyword(s):  
Intravenous Immunoglobulins ◽  
Neutralization Assay ◽  
Phage Display Library ◽  
Fusion Peptide ◽  
Significant Loss ◽  
Antibody Binding ◽  
Heptad Repeat ◽  
Minimal Loss ◽  
Neutralizing Capacity ◽  
In The Wild

Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to different levels by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 (<2-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.

scholarly journals Comprehensive characterization of the antibody responses to SARS-CoV-2 Spike protein after infection and/or vaccination

2021 ◽  
Author(s):  
Meghan E. Garrett ◽  
Jared G. Galloway ◽  
Caitlin Wolf ◽  
Jennifer K. Logue ◽  
Nicholas Franko ◽  
...  
Keyword(s):  
Immune Escape ◽  
Severe Disease ◽  
Vaccine Dose ◽  
Principal Component ◽  
Fusion Peptide ◽  
Antibody Binding ◽  
Spike Protein ◽  
Heptad Repeat ◽  
Medical Institute ◽  
Vaccine Type

ABSTRACTBackgroundControl of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal.MethodsThe epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions we determined potential escape mutations by comparing antibody binding of peptides containing wildtype residues versus peptides containing a mutant residue.ResultsIndividuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination.ConclusionsThe finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge, they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level.FundingThis work was supported by NIH grants AI138709 (PI Overbaugh) and AI146028 (PI Matsen). Julie Overbaugh received support as the Endowed Chair for Graduate Education (FHCRC). The research of Frederick Matsen was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.

scholarly journals Neutralization of ancestral SARS-CoV-2 and variants Alpha, Beta, Gamma, Delta, Zeta and Omicron by mRNA vaccination and infection-derived immunity through homologous and heterologous variants

2021 ◽  
Author(s):  
Meriem Bekliz ◽  
Kenneth Adea ◽  
Pauline Vetter ◽  
Christiane S Eberhardt ◽  
Krisztina Hosszu-Fellous ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Immune Escape ◽  
Neutralization Assay ◽  
Significant Loss ◽  
Antigenic Relationship ◽  
Double Dose ◽  
Gamma Delta ◽  
Breakthrough Infection ◽  
Neutralizing Capacity ◽  
Alpha Beta

Emerging SARS-CoV-2 variants of concern/interest (VOC/VOI) raise questions about effectiveness of neutralizing antibodies derived from infection or vaccination. As the population immunity to SARS-CoV-2 has become more complex due to prior infection and/or vaccination, understanding the antigenic relationship between variants is needed. Here, we have assessed in total 104 blood specimens from convalescent individuals after infection with early-pandemic SARS-CoV-2 (pre-VOC) or with Alpha, Beta, Gamma or Delta, post-vaccination after double-dose mRNA-vaccination and break through infections due to Delta or Omicron. Neutralization against seven authentic SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, Omicron) was assessed by plaque-reduction neutralization assay. We found highest neutralization titers against the homologous (previously infecting) variant, with lower neutralization efficiency against heterologous variants. Significant loss of neutralization for Omicron was observed but to a varying degree depending on previously infecting variant (23.0-fold in Beta-convalescence up to 56.1-fold in Alpha-convalescence), suggesting that infection-derived immunity varies, but independent of the infecting variant is only poorly protective against Omicron. Of note, Zeta VOI showed also pronounced escape from neutralization of up to 28.2-fold in Alpha convalescent samples. Antigenic mapping reveals both Zeta and Omicron as separate antigenic clusters. Double dose vaccination showed robust neutralization for Alpha, Beta, Gamma, Delta and Zeta, with fold-change reduction of only 2.8 (for Alpha) up to 6.9 (for Beta). Escape from neutralization for Zeta was largely restored in vaccinated individuals, while Omicron still showed a loss of neutralization of 85.7-fold compared to pre-VOC SARS-CoV-2. Combined immunity from infection followed by vaccination or vaccine breakthrough infection showed highest titers and most robust neutralization for heterologous variants. Breakthrough infection with Delta showed only 12.5-fold reduced neutralization for Omicron, while breakthrough infection with Omicron showed only a 1.5-fold loss for Delta, suggests that infection with antigenically different variants can boost immunity for antigens closer to the vaccine strain. Antigenic cartography showed also a tendency towards broader neutralizing capacity for heterologous variants. We conclude that the complexity of background immunity needs to be taken into account when assessing new VOCs. Development towards separate serotypes such as Zeta was already observed before Omicron emergence, thus other factors than just immune escape must contribute to Omicrons rapid dominance. However, combined infection/vaccination immunity could ultimately lead to broad neutralizing capacity also against non-homologous variants.

scholarly journals An Optimized High-Throughput Neutralization Assay for Hepatitis E Virus (HEV) Involving Detection of Secreted Porf2

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 64 ◽  
Author(s):  
Chang Liu ◽  
Wei Cai ◽  
Xin Yin ◽  
Zimin Tang ◽  
Guiping Wen ◽  
...  
Keyword(s):  
High Throughput ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Neutralization Assay ◽  
The Novel ◽  
Specific Antibodies ◽  
Neutralizing Activity ◽  
Elisa Kit ◽  
Neutralizing Capacity ◽  
Potential Threshold

Hepatitis E virus (HEV) is a common cause of acute hepatitis worldwide. Current methods for evaluating the neutralizing activity of HEV-specific antibodies include immunofluorescence focus assays (IFAs) and real-time PCR, which are insensitive and operationally complicated. Here, we developed a high-throughput neutralization assay by measuring secreted pORF2 levels using an HEV antigen enzyme-linked immunosorbent assay (ELISA) kit based on the highly replicating HEV genotype (gt) 3 strain Kernow. We evaluated the neutralizing activity of HEV-specific antibodies and the sera of vaccinated individuals (n = 15) by traditional IFA and the novel assay simultaneously. A linear regression analysis shows that there is a high degree of correlation between the two assays. Furthermore, the anti-HEV IgG levels exhibited moderate correlation with the neutralizing titers of the sera of vaccinated individuals, indicating that immunization with gt 1 can protect against gt 3 Kernow infection. We then determined specificity of the novel assay and the potential threshold of neutralizing capacity using anti-HEV IgG positive sera (n = 27) and anti-HEV IgG negative sera (n = 23). The neutralizing capacity of anti-HEV IgG positive sera was significantly stronger than that of anti-HEV IgG negative. In addition, ROC curve analysis shows that the potential threshold of neutralizing capacity of sera was 8.07, and the sensitivity and specificity of the novel assay was 88.6% and 100%, respectively. Our results suggest that the neutralization assay using the antigen ELISA kit could be a useful tool for HEV clinical research.

scholarly journals Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Raksha Das ◽  
Rohini Datta ◽  
Raghavan Varadarajan
Keyword(s):  
Cell Surface ◽  
Viral Entry ◽  
Fusion Peptide ◽  
Heptad Repeat ◽  
Subtype B ◽  
Clade C ◽  
Viral Surface ◽  
Virion Surface ◽  
Peptide Region ◽  
Hiv 1

ABSTRACT HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens. IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.

Disruption of PF4/Hep Multimolecular Complex Assembly Using a Minimally Anticoagulant Heparin (ODSH)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 723-723
Author(s):  
Manali Joglekar ◽  
Pedro Quintana ◽  
Stephen Marcus ◽  
Jian Liu ◽  
Gowthami M. Arepally
Keyword(s):  
Complex Formation ◽  
Electrostatic Interactions ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Antibody Binding ◽  
Fixed Amount ◽  
Platelet Factor ◽  
Molar Ratios

Abstract Abstract 723 Recent studies indicate that multimolecular complexes of platelet factor 4 (PF4) and heparin (H) are central to the pathogenesis of Heparin-Induced Thrombocytopenia (HIT). PF4/H multimolecular complexes are recognized preferentially by HIT antibodies (Rauova, Blood 2005) and are potently immunizing in a murine immunization model (Suvarna, Blood 2005). Because PF4/H multimolecular complexes assemble through non-specific electrostatic interactions, we hypothesized that disruption of PF4/H charge-dependent interactions could reduce immune mediated complications. To test this hypothesis, we employed a minimally anticoagulant compound (2-O, 3-O desulfated heparin, or ODSH, ParinGenix, Inc.) and characterized the charge-dependent interactions of murine PF4 (mPF4), ODSH and unfractionated heparin (UFH). In chromogenic assays of thrombin (IIa) generation, UFH was >80-fold more potent than ODSH in inactivating heparin (IC50 of residual IIa generation for UFH=3.1 nM v. ODSH= 259 nM, (Figure 1A). However, when equimolar amounts of UFH or ODSH (1.7 mM) were tested in a PF4 neutralization assay (Saggin, Thrombosis and Haemostasis 1992), the amount of mPF4 required to neutralize 50% of the anticoagulant activity of ODSH (IC50) was 25μg/mL, as compared to 73μg/mL for UFH (~3-fold difference), indicating that charge-dependent interactions, but not anticoagulant activity, were preserved between PF4 and ODSH (Figure 1B). When ODSH was added at 2.5, 5 or 10 fold molar excess to a fixed amount of UFH (6nM) in the PF4 neutralization assay, a proportionate increase in the amount of PF4 was needed to neutralize UFH, indicating that ODSH promotes the anticoagulant effect of UFH through preferential binding of PF4. To further characterize the biophysical interactions of PF4, ODSH and UFH, we used spectrophotometry and zeta potential to study the multimolecular complex formation (Suvarna, Blood 2007). We noted that mPF4 and ODSH formed multimolecular complexes at molar ratios of 2:1, whereas mPF4 and UFH complexes occurred at molar ratios of 1:1. When increasing concentrations of ODSH were added to pre-formed PF4/H multimolecular complexes, we noted a decrease in absorbance with increasing amounts of ODSH, indicating disruption of PF4/H multimolecular complexes (Figure 1C). However, when increasing amounts of UFH was added to preformed PF4/ODSH multimolecular complexes, a plateau in signal was noted, suggesting a higher affinity of ODSH for PF4. In PF4/H immunoassays, incubation of ODSH (1μg/mL) with HIT antibodies was effective in reducing antibody binding by >50% as compared to wells without ODSH. HIT antibodies did not recognize hPF4 (10mg/mL) in complex with ODSH (0.4-3.2 mg/mL), indicating minimal cross-reactivity of HIT antibodies with PF4/ODSH complexes (Figure 1D). In summary, we show that ODSH, a minimally anticoagulant heparin, can disrupt PF4/H multimolecular complex formation through charge dependent interactions and interfere with HIT antibody binding. These studies suggest that manipulation of PF4:H charge interactions can be a potential therapeutic strategy in the management of HIT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High-Dose Subcutaneous Immunoglobulins for the Treatment of Severe Treatment-Resistant Polymyositis

2014 ◽  
Vol 2014 ◽  
pp. 1-4 ◽  
Author(s):  
Cherin Patrick ◽  
Delain Jean-Christophe ◽  
Crave Jean-Charles ◽  
Cartry Odile
Keyword(s):  
Muscle Weakness ◽  
Kinase Activity ◽  
Intravenous Immunoglobulins ◽  
Immunosuppressive Drugs ◽  
Significant Loss ◽  
Creatine Kinase Activity ◽  
High Dose ◽  
Drug Resistant ◽  
Treatment Resistant ◽  
Good Patient Satisfaction

Polymyositis is a rare debilitating condition characterized by chronic inflammation and muscle weakness. Standard treatments include corticosteroids and immunosuppressants; however, resistance to these regimens may develop. Intravenous immunoglobulins (IVIg) are thus recommended for patients with drug-resistant polymyositis. The patient presented a resistant polymyositis with severe muscle weakness, increasing dysphagia, and significant loss in weight. Subcutaneous immunoglobulins (SCIg) were initiated after failure of steroids and immunosuppressive drugs. SCIg was given twice per week (2 then 1.3 g/kg/month). Clinical recovery was observed within 2 months after the SCIg initiation. After several injections, the patient showed a progressive improvement in muscle strength. Serum creatine kinase activity decreased to normal levels, and dysphagia was resolved. The SC injections were generally well tolerated and good patient satisfaction was reported. This promising observation suggests that SCIg may be useful in active and refractory polymyositis.

scholarly journals Inhibition of Human Coronavirus NL63 Infection at Early Stages of the Replication Cycle

2006 ◽  
Vol 50 (6) ◽  
pp. 2000-2008 ◽  
Author(s):  
Krzysztof Pyrc ◽  
Berend Jan Bosch ◽  
Ben Berkhout ◽  
Maarten F. Jebbink ◽  
Ronald Dijkman ◽  
...  
Keyword(s):  
Respiratory Illness ◽  
Intravenous Immunoglobulins ◽  
Replication Cycle ◽  
Heptad Repeat ◽  
Effective Vaccine ◽  
Cellular Receptor ◽  
Acute Respiratory Illness ◽  
Human Coronavirus ◽  
Synthetic Compounds ◽  
Multiple Stages

ABSTRACT Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in young children and elderly and immunocompromised persons. Further analysis of HCoV-NL63 pathogenicity seems warranted, in particular because the virus uses the same cellular receptor as severe acute respiratory syndrome-associated coronavirus. As there is currently no HCoV-NL63-specific and effective vaccine or drug therapy available, we evaluated several existing antiviral drugs and new synthetic compounds as inhibitors of HCoV-NL63, targeting multiple stages of the replication cycle. Of the 28 compounds that we tested, 6 potently inhibited HCoV-NL63 at early steps of the replication cycle. Intravenous immunoglobulins, heptad repeat 2 peptide, small interfering RNA1 (siRNA1), siRNA2, β-d -N 4-hydroxycytidine, and 6-azauridine showed 50% inhibitory concentrations of 125 μg/ml, 2 μM, 5 nM, 3 nM, 400 nM, and 32 nM, respectively, and low 50% cytotoxicity concentrations (>10 mg/ml, >40 μM, >200 nM, >200 nM, >100 μM, and 80 μM, respectively). These agents may be investigated further for the treatment of coronavirus infections.

scholarly journals A review of the impacts of salmonid farming on marine coastal ecosystems in the southeast Pacific

2006 ◽  
Vol 63 (7) ◽  
pp. 1338-1345 ◽  
Author(s):  
Alejandro H. Buschmann ◽  
Verónica A. Riquelme ◽  
María C. Hernández-González ◽  
Daniel Varela ◽  
Jaime E. Jiménez ◽  
...  
Keyword(s):  
Native Species ◽  
Chemical Properties ◽  
Ecosystem Approach ◽  
Coastal Ecosystems ◽  
Significant Loss ◽  
Current Evidence ◽  
Farmed Fish ◽  
Physico Chemical ◽  
In The Wild ◽  
Farming Industry

Abstract The production of farmed salmonids in Chile reached 550 000 t in 2004. The industry is considered to be consolidated, but with potential for further expansion to the south into pristine coastal areas. The environmental impacts of the salmonid farming industry in Chile were reviewed in 1996, and evidence at that time did not suggest significant adverse effects. However, after almost ten years of sustained growth, current evidence indicates that significant loss of benthic biodiversity and localized changes in the physico-chemical properties of sediments have occurred in areas with salmonid farms. Furthermore, the presence of these farms significantly increases in pulses the density of dinoflagellates. Data suggest that escaped farmed fish may have an impact on native species, although their survival in the wild appears low. The abundance of omnivorous diving and carrion-feeding marine birds increased from twofold to fivefold in areas with salmon farms compared with control areas without them. It is urgent that an ecosystem approach be implemented to assess all impacts of salmonid farming on coastal ecosystems in southern Chile.

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