30 research outputs found
Evaluation of a commercial ELISA as alternative to plaque reduction neutralization test to detect neutralizing antibodies against SARS-CoV-2
High-throughput detection of neutralizing antibodies against SARS-CoV-2 presents a valuable tool for vaccine trials or investigations of population immunity. We evaluate the performance of the first commercial surrogate virus neutralization test (sVNT, GenScript Biotech) against SARS-CoV-2 plaque reduction neutralization test (PRNT) in convalescent and vaccinated individuals. We compare it to five other ELISAs, two of which are designed to detect neutralizing antibodies. In 491 pre-vaccination serum samples, sVNT missed 23.6% of PRNT-positive samples when using the manufacturer-recommended cutoff of 30% binding inhibition. Introducing an equivocal area between 15 and 35% maximized sensitivity and specificity against PRNT to 72.8–93.1% and 73.5–97.6%, respectively. The overall diagnostic performance of the other ELISAs for neutralizing antibodies was below that of sVNT. Vaccinated individuals exhibited higher antibody titers by PRNT (median 119.8, IQR 56.7–160) and binding inhibition by sVNT (median 95.7, IQR 88.1–96.8) than convalescent patients (median 49.1, IQR 20–62; median 52.9, IQR 31.2–76.2). GenScript sVNT is suitable to screen for SARS-CoV-2-neutralizing antibodies; however, to obtain accurate results, confirmatory testing by PRNT in a equivocal area is required. This equivocal area may require adaptation for use in vaccinated individuals, due to higher antibody titers.Peer Reviewe
Amantadine Inhibits SARS-CoV-2 In Vitro
Since the SARS-CoV-2 pandemic started in late 2019, the search for protective vaccines and for drug treatments has become mandatory to fight the global health emergency. Travel restrictions,social distancing, and face masks are suitable counter measures, but may not bring the pandemic under control because people will inadvertently or at a certain degree of restriction severity or duration become incompliant with the regulations. Even if vaccines are approved, the need for antiviral agents against SARS-CoV-2 will persist. However, unequivocal evidence for efficacy againstSARS-CoV-2 has not been demonstrated for any of the repurposed antiviral drugs so far. Amantadinewas approved as an antiviral drug against influenza A, and antiviral activity against SARS-CoV-2has been reasoned by analogy but without data. We tested the efficacy of amantadine in vitro in Vero E6 cells infected with SARS-CoV-2. Indeed, amantadine inhibited SARS-CoV-2 replication in two separate experiments with IC50 concentrations between 83 and 119ÎĽM. Although these IC50 concentrations are above therapeutic amantadine levels after systemic administration, topical admin-istration by inhalation or intranasal instillation may result in sufficient amantadine concentration in the airway epithelium without high systemic exposure. However, further studies in other models are needed to prove this hypothesis.Peer Reviewe
Longitudinal SARS-CoV-2 seroepidemiological investigation among healthcare workers at a tertiary care hospital in Germany
Background:
SARS-CoV-2 cases in Germany increased in early March 2020. By April 2020, cases among health care workers (HCW) were detected across departments at a tertiary care hospital in Berlin, prompting a longitudinal investigation to assess HCW SARS-CoV-2 serostatus with an improved testing strategy and associated risk factors.
Methods:
In May/June and December 2020, HCWs voluntarily provided blood for serology and nasopharyngeal/oropharyngeal (NP/OP) samples for real-time polymerase chain reaction (PCR) and completed a questionnaire. A four-tiered SARS-CoV-2 serological testing strategy including two different enzyme-linked immunosorbent assays (ELISA) and biological neutralization test (NT) was used. ELISA-NT correlation was assessed using Pearson’s correlation coefficient. Sociodemographic and occupational factors associated with seropositivity were assessed with multivariate logistic regression.
Results:
In May/June, 18/1477 (1.2%) HCWs were SARS-CoV-2 seropositive, followed by 56/1223 (4.6%) in December. Among those tested in both, all seropositive in May/June remained seropositive by ELISA and positive by NT after 6 months. ELISA ratios correlated well with NT titres in May/June (R = 0.79) but less so in December (R = 0.41). Those seropositive reporting a past SARS-CoV-2 positive PCR result increased from 44.4% in May/June to 85.7% in December. HCWs with higher occupational risk (based on profession and working site), nurses, males, and those self-reporting COVID-19-like symptoms had significantly higher odds of seropositivity.
Conclusions:
This investigation provides insight into the burden of HCW infection in this local outbreak context and the antibody dynamics over time with an improved robust testing strategy. It also highlights the continued need for effective infection control measures particularly among HCWs with higher occupational risk.Peer Reviewe
Primary ChAdOx1 vaccination does not reactivate pre-existing, cross-reactive immunity
Currently available COVID-19 vaccines include inactivated virus, live attenuated virus, mRNA-based, viral vectored and adjuvanted protein-subunit-based vaccines. All of them contain the spike glycoprotein as the main immunogen and result in reduced disease severity upon SARS-CoV-2 infection. While we and others have shown that mRNA-based vaccination reactivates pre-existing, cross-reactive immunity, the effect of vector vaccines in this regard is unknown. Here, we studied cellular and humoral responses in heterologous adenovirus-vector-based ChAdOx1 nCOV-19 (AZ; Vaxzeria, AstraZeneca) and mRNA-based BNT162b2 (BNT; Comirnaty, BioNTech/Pfizer) vaccination and compared it to a homologous BNT vaccination regimen. AZ primary vaccination did not lead to measurable reactivation of cross-reactive cellular and humoral immunity compared to BNT primary vaccination. Moreover, humoral immunity induced by primary vaccination with AZ displayed differences in linear spike peptide epitope coverage and a lack of anti-S2 IgG antibodies. Contrary to primary AZ vaccination, secondary vaccination with BNT reactivated pre-existing, cross-reactive immunity, comparable to homologous primary and secondary mRNA vaccination. While induced anti-S1 IgG antibody titers were higher after heterologous vaccination, induced CD4(+) T cell responses were highest in homologous vaccinated. However, the overall TCR repertoire breadth was comparable between heterologous AZ-BNT-vaccinated and homologous BNT-BNT-vaccinated individuals, matching TCR repertoire breadths after SARS-CoV-2 infection, too. The reasons why AZ and BNT primary vaccination elicits different immune response patterns to essentially the same antigen, and the associated benefits and risks, need further investigation to inform vaccine and vaccination schedule development
Untersuchung und Eindämmung eines SARS-CoV-2-Alpha-Ausbruchs in einer Pflegeeinrichtung im Landkreis Dithmarschen, Juni 2021
Bewohnende und Personal in Pflegeeinrichtungen gehören zu den Personengruppen, die ab dem 27.12.2020 in Deutschland priorisiert die COVID-19-Impfung erhielten. Diese zeigte eine gute Wirksamkeit hinsichtlich des Schutzes vor Infektionen mit der Alpha-Variante und eine sehr hohe Wirksamkeit vor schweren Krankheitsverläufen. In einer Pflegeeinrichtung im Landkreis Dithmarschen kam es zwischen dem 01.04. und 23.06.2021 dennoch zu einem SARS-CoV-2-Ausbruch, bei dem eine hohe Anzahl vollständig geimpfter Personen infiziert wurde und teilweise schwer erkrankte oder verstarb. Beschrieben werden zum einen die Methoden, die bei der Ausbruchsuntersuchung im Pflegeheim angewandt wurden, und zum anderen die Maßnahmen, die schlussendlich zur Ausbruchseindämmung beitrugen.Peer Reviewe
Multicountry study of SARS-CoV-2 and associated risk factors among healthcare workers in Cote d’Ivoire, Burkina Faso and South Africa
DATA AVAILABILITY : The data sets generated for this study are available upon request from the corresponding author.BACKGROUND : Reports on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread across Africa have varied, including among healthcare workers (HCWs). This study assessed the comparative SARS-CoV-2 burden and associated risk factors among HCWs in three African countries. METHODS : A multicentre study was conducted at regional healthcare facilities in Côte d’Ivoire (CIV), Burkina Faso (BF) and South Africa (SA) from February to May 2021. HCWs provided blood samples for SARS-CoV-2 serology and nasopharyngeal/oropharyngeal swabs for testing of acute infection by polymerase chain reaction and com- pleted a questionnaire. Factors associated with seropositivity were assessed with logistic regression. RESULTS : Among 719 HCWs, SARS-CoV-2 seroprevalence was 34.6% (95% confidence interval 31.2 to 38.2), rang- ing from 19.2% in CIV to 45.7% in BF. A total of 20 of 523 (3.8%) were positive for acute SARS-CoV-2 infection. Female HCWs had higher odds of SARS-CoV-2 seropositivity compared with males, and nursing staff, allied health professionals, non-caregiver personnel and administration had higher odds compared with physicians. HCWs also reported infection prevention and control (IPC) gaps, including 38.7% and 29% having access to respirators and IPC training, respectively, in the last year. CONCLUSIONS : This study was a unique comparative HCW SARS-CoV-2 investigation in Africa. Seroprevalence estimates varied, highlighting distinctive population/facility-level factors affecting COVID-19 burden and the importance of established IPC programmes to protect HCWs and patients.The German Federal Ministry of Education and Research Programme for Research Networks for Health Innovations in Sub-Saharan Africa (ANDEMIA), the German Federal Ministry of Health Global Health Protection Programme (ARGOS project) and the National Robert Koch Institute in Germany.https://academic.oup.com/trstmham2023Medical MicrobiologyMedical VirologySDG-03:Good heatlh and well-bein
Aufklärung orthopockenviraler Adaptionsmechanismen mittels Proteomik und Genomik
Adaptive changes of viruses enable the virus-host coevolution and infection of new host species. While viruses with an RNA genome adapt primarily by genomic variabilities, adaptation mechanisms of viruses with a DNA genome have remained largely elusive. Since genomes of DNA viruses are more stable compared to genomes of RNA viruses, it is assumed that mechanisms other than genomic changes underlie the adaptation of DNA viruses. However, these mechanisms have hardly been investigated so far. Since isolated virus particles are capable of crossing the species barrier, it is assumed that adaptive changes can be identified in the virus particles themselves. Therefore, this study aimed to elucidate adaptation mechanisms of DNA virus particles by proteomics and genomics technologies. While next-generation sequencing technologies are frequently used to analyze genomic changes of viruses, adaptive changes of virus particles by mass spectrometry-based proteomics have not been analyzed yet.
In the present study, cowpox virus (CPXV) was used as a model virus and cell culture as a model system to study adaptive changes of DNA virus particles. CPXV is a member of the genus Orthopoxvirus (OPV) and is able infect a remarkably broad range of host species, e.g. rats, cats, elephants and humans. Increasing numbers of CPXV infections in Europe underline the need for a comprehensive understanding of OPV adaptation mechanisms.
CPXV particles were isolated from a rat, which is a natural host of these viruses, and serially passaged five times in a rat and a human cell line. During passaging, an increase in viral fitness was observed exclusively in human cells, suggesting an adaptation of virus particles. Strikingly, proteome analysis revealed that the composition of virus particles changed in a cell line-specific manner, while the viral genome remained overall stable during passaging in both cell lines. Because several ubiquitination sites in virus proteins were identified, the role of ubiquitin for CPXV infection was analyzed. It was shown by the first global ubiquitination site analysis of virus particles that ubiquitin is a major conserved CPXV modification. Additionally, the dependence of CPXV replication on this protein modification was verified, making ubiquitination changes an attractive hypothesis of OPV adaptation. Furthermore, it was shown that CPXV particles incorporate intact transcripts, which presumably enable the rapid expression of viral immunomodulatory proteins upon infection.
Summarized, the results of the present study lead to new findings about OPV adaptation mechanisms in vitro. These mechanisms may also apply to in vivo adaptation of DNA viruses and may enable, for example, a crossing of the species barrier. The methods established in this study enable the further characterization of OPV adaptation and, moreover, can be applied to elucidate adaptation mechanisms of viruses belonging to other families.Adaptive Veränderungen von Viren ermöglichen eine Virus-Wirt Koevolution und die Infektion neuer Spezies. Während der Adaption von Viren mit RNA Genom primär eine genomische Variabilität zugrunde liegt, sind Adaptationsmechanismen von Viren mit DNA Genom bislang nur wenig erforscht. Da das Genom von DNA-Viren stabiler ist als das von RNA-Viren, wird vermutet, dass der Adaptation von DNA-Viren andere Mechanismen zugrunde liegen als rein genomische Veränderungen. Weiterhin ist anzunehmen, dass adaptive Veränderungen im Viruspartikel selbst zu identifizieren sind, da isolierte Viruspartikel in der Lage sind die Speziesbarriere zu überwinden. Das Ziel der hier vorliegenden Studie war es daher Adaptationsmechanismen von DNA-Viren mittels Proteomik und Genomik aufzuklären. Während genomische Veränderungen von Viren häufig mittels Next Generation Sequencing analysiert werden, wurden proteomische Veränderungen viraler Partikel bislang noch nicht mit Massenspektrometrie-basierten Methoden analysiert.
Als Modell-DNA-Virus für die hier vorliegende Studie wurde das Kuhpockenvirus (CPXV) gewählt und Zellkultur als System um adaptive Veränderungen zu untersuchen. CPXV gehören zum Genus der Orthopockenviren (OPV) und können eine Vielzahl von Spezies infizieren, z.B. Ratten, Katzen, Elefanten und Menschen. Die Anzahl an CPXV-Infektionen in Europa nimmt stetig zu, weshalb es notwendig ist Adaptionsmechanismen von OPV aufzudecken.
CPXV Partikel wurden aus einer Ratte isoliert und für fünf Passagen in einer humanen und in einer Ratten Zelllinie vermehrt. Dabei konnte ausschließlich in humanen Zellen eine Steigerung der viralen Fitness beobachtet werden, was auf eine Adaptation der Viren hindeutet. Die Proteomanalyse zeigte, dass sich die Viruspartikel während des Passagierens Zelllinien-spezifisch veränderten, während das virale Genom jedoch insgesamt stabil blieb. Da mehrere Ubiquitinierungsstellen in viralen Proteinen detektiert wurden, wurde die Rolle von Ubiquitin für die CPXV Infektion untersucht. Mittels der ersten globalen Ubiquitinomanalyse von Viruspartikeln konnte gezeigt werden, dass Ubiquitin eine konservierte Modifikation in CPXV Proteinen ist, dessen biologische Relevanz in weiteren Experimenten bestätigt werden konnte. Veränderungen der Ubiquitinierung stellen somit eine attraktive Hypothese zur Adaptation von OPV dar. Darüber hinaus konnte gezeigt werden, dass CPXV Partikel Transkripte inkorporieren, die vermutlich eine schnelle Expression viraler Immunmodulatoren ermöglichen.
Die Ergebnisse der vorliegenden Studie führen zu neuen Erkenntnissen über die Adaptationsmechanismen von OPV in vitro, die möglicherweise auch für die Adaption von DNA-Viren in vivo gelten. Die im Rahmen dieser Arbeit etablierten Methoden können weiterhin dazu genutzt werden, um die Adaptation von OPV näher zu charakterisieren und um die Adaptation von Viren anderer Familien zu untersuchen
Elucidation of Orthopoxvirus Adaptation Mechanisms by Proteomics and Genomics
Adaptive changes of viruses enable the virus-host coevolution and infection of new host species. While viruses with an RNA genome adapt primarily by genomic variabilities, adaptation mechanisms of viruses with a DNA genome have remained largely elusive. Since genomes of DNA viruses are more stable compared to genomes of RNA viruses, it is assumed that mechanisms other than genomic changes underlie the adaptation of DNA viruses. However, these mechanisms have hardly been investigated so far. Since isolated virus particles are capable of crossing the species barrier, it is assumed that adaptive changes can be identified in the virus particles themselves. Therefore, this study aimed to elucidate adaptation mechanisms of DNA virus particles by proteomics and genomics technologies. While next-generation sequencing technologies are frequently used to analyze genomic changes of viruses, adaptive changes of virus particles by mass spectrometry-based proteomics have not been analyzed yet. In the present study, cowpox virus (CPXV) was used as a model virus and cell culture as a model system to study adaptive changes of DNA virus particles. CPXV is a member of the genus Orthopoxvirus (OPV) and is able infect a remarkably broad range of host species, e.g. rats, cats, elephants and humans. Increasing numbers of CPXV infections in Europe underline the need for a comprehensive understanding of OPV adaptation mechanisms. CPXV particles were isolated from a rat, which is a natural host of these viruses, and serially passaged five times in a rat and a human cell line. During passaging, an increase in viral fitness was observed exclusively in human cells, suggesting an adaptation of virus particles. Strikingly, proteome analysis revealed that the composition of virus particles changed in a cell line-specific manner, while the viral genome remained overall stable during passaging in both cell lines. Because several ubiquitination sites in virus proteins were identified, the role of ubiquitin for CPXV infection was analyzed. It was shown by the first global ubiquitination site analysis of virus particles that ubiquitin is a major conserved CPXV modification. Additionally, the dependence of CPXV replication on this protein modification was verified, making ubiquitination changes an attractive hypothesis of OPV adaptation. Furthermore, it was shown that CPXV particles incorporate intact transcripts, which presumably enable the rapid expression of viral immunomodulatory proteins upon infection. Summarized, the results of the present study lead to new findings about OPV adaptation mechanisms in vitro. These mechanisms may also apply to in vivo adaptation of DNA viruses and may enable, for example, a crossing of the species barrier. The methods established in this study enable the further characterization of OPV adaptation and, moreover, can be applied to elucidate adaptation mechanisms of viruses belonging to other families
Application of spectral library prediction for parallel reaction monitoring of viral peptides
A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future.Peer Reviewe
Combined Proteomics/Genomics Approach Reveals Proteomic Changes of Mature Virions as a Novel Poxvirus Adaptation Mechanism
DNA viruses, like poxviruses, possess a highly stable genome, suggesting that adaptation of virus particles to specific cell types is not restricted to genomic changes. Cowpox viruses are zoonotic poxviruses with an extraordinarily broad host range, demonstrating their adaptive potential in vivo. To elucidate adaptation mechanisms of poxviruses, we isolated cowpox virus particles from a rat and passaged them five times in a human and a rat cell line. Subsequently, we analyzed the proteome and genome of the non-passaged virions and each passage. While the overall viral genome sequence was stable during passaging, proteomics revealed multiple changes in the virion composition. Interestingly, an increased viral fitness in human cells was observed in the presence of increased immunomodulatory protein amounts. As the only minor variant with increasing frequency during passaging was located in a viral RNA polymerase subunit and, moreover, most minor variants were found in transcription-associated genes, protein amounts were presumably regulated at transcription level. This study is the first comparative proteome analysis of virus particles before and after cell culture propagation, revealing proteomic changes as a novel poxvirus adaptation mechanism