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Scientific Reports Feb 2019Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2)...
Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2) viruses hinder traditional antigenic characterization using haemagglutination inhibition and promote selection of HA mutants. Thus, alternative approaches are needed to reliably assess antigenic relatedness between circulating viruses and vaccines. We developed a high content imaging-based neutralization test (HINT) to reduce antigenic mischaracterization resulting from virus adaptation to cell culture. Ferret reference antisera were raised using clinical specimens containing viruses representing recent vaccine strains. Analysis of viruses circulating during 2011-2018 showed that gain of an N158-linked glycosylation in HA was a molecular determinant of antigenic distancing between A/Hong Kong/4801/2014-like (clade 3C.2a) and A/Texas/50/2012-like viruses (clade 3C.1), while multiple evolutionary HA F193S substitution were linked to antigenic distancing from A/Switzerland/97152963/2013-like (clade 3C.3a) and further antigenic distancing from A/Texas/50/2012-like viruses. Additionally, a few viruses carrying HA T135K and/or I192T showed reduced neutralization by A/Hong Kong/4801/2014-like antiserum. Notably, this technique elucidated the antigenic characteristics of clinical specimens, enabling direct characterization of viruses produced in vivo, and eliminating in vitro culture, which rapidly alters the genotype/phenotype. HINT is a valuable new antigenic analysis tool for vaccine strain selection.
Topics: Animals; Antibodies, Viral; Antigens, Viral; Ferrets; Glycosylation; Hemagglutination Inhibition Tests; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Immune Sera; Influenza A Virus, H3N2 Subtype; Influenza Vaccines; Influenza, Human; Neutralization Tests; Phylogeny
PubMed: 30804469
DOI: 10.1038/s41598-019-39276-1 -
Methods in Molecular Biology (Clifton,... 2015Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for...
Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody.
Topics: Animals; Antibodies, Monoclonal; Ascites; Bacterial Proteins; Chromatography, Affinity; Chromatography, Liquid; Culture Media, Conditioned; Humans; Hybridomas; Immobilized Proteins; Immune Sera; Immunoglobulin Isotypes; Mice; Protein Binding; Staphylococcal Protein A
PubMed: 26160561
DOI: 10.1007/978-1-4939-2742-5_3 -
Marine Drugs Aug 2017The peer-reviewed marine pharmacology literature from 2012 to 2013 was systematically reviewed, consistent with the 1998-2011 reviews of this series. Marine pharmacology... (Review)
Review
Marine Pharmacology in 2012-2013: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis, and Antiviral Activities; Affecting the Immune and Nervous Systems, and Other Miscellaneous Mechanisms of Action.
The peer-reviewed marine pharmacology literature from 2012 to 2013 was systematically reviewed, consistent with the 1998-2011 reviews of this series. Marine pharmacology research from 2012 to 2013, conducted by scientists from 42 countries in addition to the United States, reported findings on the preclinical pharmacology of 257 marine compounds. The preclinical pharmacology of compounds isolated from marine organisms revealed antibacterial, antifungal, antiprotozoal, antituberculosis, antiviral and anthelmitic pharmacological activities for 113 marine natural products. In addition, 75 marine compounds were reported to have antidiabetic and anti-inflammatory activities and affect the immune and nervous system. Finally, 69 marine compounds were shown to display miscellaneous mechanisms of action which could contribute to novel pharmacological classes. Thus, in 2012-2013, the marine natural product pharmacology pipeline provided novel pharmacology and lead compounds to the marine pharmaceutical pipeline, and contributed significantly to potentially novel therapeutic approaches to several global disease categories.
Topics: Anti-Inflammatory Agents; Anticoagulants; Antifungal Agents; Antiprotozoal Agents; Antitubercular Agents; Antiviral Agents; Aquatic Organisms; Biological Products; Humans; Hypoglycemic Agents; Immune Sera; Marine Biology; Nervous System
PubMed: 28850074
DOI: 10.3390/md15090273 -
Infection, Genetics and Evolution :... Mar 2019Here we report studies of the antigenic relationship of West Nile virus (WNV) and Usutu virus (USUV), two zoonotic flaviviruses from Italy, together with a Japanese...
Here we report studies of the antigenic relationship of West Nile virus (WNV) and Usutu virus (USUV), two zoonotic flaviviruses from Italy, together with a Japanese encephalitis virus (JEV) strain and compared them with their genetic relationship using the immunodominant viral E protein. Thirty-nine isolates and reference strains were inactivated and used to immunize rabbits to produce hyper immune sera. Serum samples were tested by neutralization against all isolates and results visualized by generating antigenic map. Strains of WNV, USUV, and JEV grouped in separate clusters on the antigenic map. JEV was closer antigenically to USUV (mean of 3.5 Antigenic Unit, AU, equivalent to a 2-fold change in antibody titer) than to WNV strains (mean of 6 AU). A linear regression model predicted, on average, one unit of antigenic change, equivalent to a 2-fold change in antibody titer, for every 22 amino acid substitutions in the E protein ectodomain. Overall, antigenic map was demonstrated to be robust and consistent with phylogeny of the E protein. Indeed, the map provided a reliable means of visualizing and quantifying the relationship between these flaviviruses. Further antigenic analyses employing representative strains of extant serocomplexes are currently underway. This will provide a more in deep knowledge of antigenic relationships between flaviviruses.
Topics: Amino Acid Sequence; Animals; Antibodies, Neutralizing; Antibodies, Viral; Antigens, Viral; Flavivirus; Flavivirus Infections; Immune Sera; Italy; Phylogeny; Serologic Tests; Zoonoses
PubMed: 30517880
DOI: 10.1016/j.meegid.2018.11.023 -
Viruses Feb 2022In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of... (Comparative Study)
Comparative Study
In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen-antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera-as in cases of non-ARDS sera-combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen-antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.
Topics: Adult; Aged; Aged, 80 and over; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Convalescence; Enzyme-Linked Immunosorbent Assay; Female; Hospitalization; Humans; Immune Sera; Immunity, Humoral; Male; Middle Aged; Neutralization Tests; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 35216003
DOI: 10.3390/v14020410 -
Journal of Medical Virology Apr 2023To investigate the antigenic changes in parechovirus 1 (PeVA1), seroepidemiological analyses were performed against the Harris strain (Harris), isolated in 1956, and...
To investigate the antigenic changes in parechovirus 1 (PeVA1), seroepidemiological analyses were performed against the Harris strain (Harris), isolated in 1956, and PeVA1/Yamagata.JPN/2021-4785, isolated in 2021, using immune sera and 207 and 237 human serum specimens collected in 2021 and 1976, respectively. Although rabbit immune sera showed the highest neutralization antibody (NT-Ab) titers against the immunized viruses at 1:12 800-1:102 400, they were cross-reactive at 1:400-1:800. All 62 Yamagata isolates obtained between 2001 and 2021 (Yamagata strains), belonging to phylogenetic lineage 1B, reacted more strongly (mostly 4-64 times) to antiserum against PeVA1/Yamagata.JPN/2021-4785 than to antiserum against Harris, belonging to phylogenetic lineage 1 A. Human serum specimens obtained in 2021 showed higher NT-Ab titers against PeVA1/Yamagata.JPN/2021-4785, whereas those obtained in 1976 had similar NT-Ab titers against both strains. These findings suggested that Yamagata strains and Harris were antigenically cross-reactive, although there were differences. There are still high NT-Abs titers present against Harris in 2021 in particular, indicating that PeVA1 has been in circulation with high immunity in the population. In conclusion, this study suggested that PeVA1 has been endemically perpetuated with only minor antigenic changes as well as with high immunity over several decades in the community.
Topics: Animals; Humans; Rabbits; Parechovirus; Japan; Phylogeny; Viruses; Immune Sera; Influenza, Human
PubMed: 36951317
DOI: 10.1002/jmv.28696 -
International Journal of Molecular... May 2023Envenomation by venomous fish, although not always fatal, is capable of causing damage to homeostasis by activating the inflammatory process, with the formation of...
Envenomation by venomous fish, although not always fatal, is capable of causing damage to homeostasis by activating the inflammatory process, with the formation of edema, excruciating pain, necrosis that is difficult to heal, as well as hemodynamic and cardiorespiratory changes. Despite the wide variety of pharmacological treatments used to manage acute symptoms, none are effective in controlling envenomation. Knowing the essential role of neutralizing polyclonal antibodies in the treatment of envenoming for other species, such as snakes, this work aimed to produce a polyclonal antiserum in mice and test its ability to neutralize the main toxic effects induced by the venoms of the main venomous Brazilian fish. We found that the antiserum recognizes the main toxins present in the different venoms of , , , and and was effective in pre-incubation trials. In an independent test, the antiserum applied immediately to the topical application of , , and venoms completely abolished the toxic effects on the microcirculation, preventing alterations such as arteriolar contraction, slowing of blood flow in postcapillary venules, venular stasis, myofibrillar hypercontraction, and increased leukocyte rolling and adherence. The edematogenic and nociceptive activities induced by these venoms were also neutralized by the immediate application of the antiserum. Importantly, the antiserum prevented the acute inflammatory response in the lungs induced by the venom. The success of antiserum containing neutralizing polyclonal antibodies in controlling the toxic effects induced by different venoms offers a new strategy for the treatment of fish envenomation in Brazil.
Topics: Mice; Animals; Fish Venoms; Immune Sera; Batrachoidiformes; Perciformes; Catfishes
PubMed: 37176045
DOI: 10.3390/ijms24098338 -
Cancer Treatment and Research... 2021Covid-19 Pneumonia of SARS-CoV-2 pandemic infection, persists to have high disease burden especially in cancer patients. Increased inflammation and thromboembolic...
Covid-19 Pneumonia of SARS-CoV-2 pandemic infection, persists to have high disease burden especially in cancer patients. Increased inflammation and thromboembolic processes are blamed to influence cancer patients more than the others but due to lack of knowledge regarding the pathophysiology of the both the virus itself and the response of the host, more basic and translational disease modeling research is needed to understand Cancer-Covid-19 interaction. In this study, serum samples from the patients, who were hospitalized due to Covid-19 pneumonia, applied to different cancer cells and cytotoxicity, motility, proliferation and gene expression analysis were performed. Serum samples derived from healthy volunteers and the fetal bovine serum that is used regularly in cell culture experiments used as controls. Hospitalized Covid-19 patients who had also cancer, were retrospectively screened, and their clinical course were recorded. Overall 12 Patient (PS) and 4 healthy serums (CS) were included in the experiments. PS applied cells showed increased motility in A549 cells as well as lost cell to cell connection in MCF7 and HCT116 cells, and induced expression of VIM, ZEB1 and SNAIL2 mRNA levels. Eight cancer diagnosed patients who were hospitalized due to Covid-19 between April and September 2020 were also reviewed retrospectively, which 5 of them were dead during SARS-CoV-2 infection. Thorax CT images of the 2 patients showed increased metastatic nodules in the lungs as of January 2021. The results of the study indicate that metastasis may be one of the prolonged consequences of COVID-19 pandemic in cancer sufferers.
Topics: Adult; Aged; COVID-19; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytotoxicity, Immunologic; Epithelial-Mesenchymal Transition; Female; Humans; Immune Sera; Lung Neoplasms; Male; Middle Aged; Neoplasms
PubMed: 34090218
DOI: 10.1016/j.ctarc.2021.100406 -
The Lancet. Microbe Oct 2021
Topics: COVID-19; Humans; Immune Sera; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 34458880
DOI: 10.1016/S2666-5247(21)00217-2 -
Transfusion Nov 2022Four amino acids are involved in epitope formation of human neutrophil antigens (HNA)-1 alleles, located at positions 36, 65, 78, and 82. HNA-1a and HNA-1b alloantibody...
BACKGROUND
Four amino acids are involved in epitope formation of human neutrophil antigens (HNA)-1 alleles, located at positions 36, 65, 78, and 82. HNA-1a and HNA-1b alloantibody epitopes were recently characterized. The HNA-1b allele also carries the HNA-1d epitope p.78A&p.82N. The current study aimed to identify compound antibody specificities in HNA-1b alloantisera, especially the presence of anti-HNA-1d.
STUDY DESIGN AND METHODS
For investigation of binding epitopes for HNA-1b alloantibodies, cells stably expressing different HNA-1 alleles were generated and tested against previously well-characterized HNA-1b antisera (n = 11) in an antigen capture assay. Sera with p.82N specificity or p.36S and p.82N specificity were additionally analyzed using adsorption and elution methods.
RESULTS
Three amino acids, p.36S, p.78A, and p.82N, are involved in epitope formation of HNA-1b. The following specificities were identified in 11 HNA-1b alloantisera: p.36S (6/11), p.82N (9/11), and p.78A&p.82N (8/11), of which p.36S was identified as a sole entity in 2/11, whereas 9/11 antisera contained a polyspecific mixture of anti-p.36S, p.82N (1/11), and anti-p.78A&p.82N in combination with anti p.82N (5/11) or compound specificities of anti-p.36S, p.82N, and p.78A&p82N (3/11). In seven of eight antisera with p.82N specificity, anti-p.78A&p.82N was detected.
DISCUSSION
Analysis of HNA-1b antisera indicates compound specificities for HNA-1b alloantibodies with a high variation between HNA-1b immunized individuals. Amino acids p.36S, p.82N, and p.78A&p.82N are necessary for HNA-1b epitope formation. The HNA-1d epitope is recognized by 73% (8/11) of HNA-1b immunized individuals.
Topics: Humans; Antibody Specificity; Isoantigens; Neutrophils; Isoantibodies; Epitopes; Immune Sera; Amino Acids
PubMed: 36173690
DOI: 10.1111/trf.17118