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Continuously emerging variants of concern (VOCs) sustain the SARS-CoV-2 pandemic. The SARS-CoV-2 Omicron/B.1.1.529 VOC harbours multiple mutations in the spike protein associated with high infectivity and efficient evasion from humoral immunity induced by previous infection or vaccination. By performing in-depth comparisons of the SARS-CoV-2-specific T-cell epitope repertoire after infection and messenger RNA vaccination, we demonstrate that spike-derived epitopes were not dominantly targeted in convalescent individuals compared to non-spike epitopes. In vaccinees, however, we detected a broader spike-specific T-cell response compared to convalescent individuals. Booster vaccination increased the breadth of the spike-specific T-cell response in convalescent individuals but not in vaccinees with complete initial vaccination. In convalescent individuals and vaccinees, the targeted T-cell epitopes were broadly conserved between wild-type SARS-CoV-2 variant B and Omicron/B.1.1.529. Hence, our data emphasize the relevance of vaccine-induced spike-specific CD8+ T-cell responses in combating VOCs including Omicron/B.1.1.529 and support the benefit of boosting convalescent individuals with mRNA vaccines.

Was ist neu? Prävalenz und Risikofaktoren Die Gesamthäufigkeit von Langzeitbeschwerden ist unklar. Als Risikofaktoren konnten bisher der Schweregrad der Akuterkrankung, weibliches Geschlecht und Grunderkrankungen identifiziert werden. Klinik und Pathogenese Die Pathogenese ist nicht geklärt. Ebenso unbekannt ist die Prognose über den Verlauf der Beschwerden. Wichtig erscheint ein symptomgeleiteter Ausschluss von behandlungsbedürftigen Alternativdiagnosen. Für den Beschwerdekomplex gibt es bislang weder einen einheitlichen Begriff („Long-COVID“) noch eine einheitliche Definition. Häufig wird die Definition des NICE verwendet, das zwischen anhaltendem COVID-19 bei Beschwerden 4–12 Wochen bzw. einem Post-COVID-19-Syndrom mehr als 12 Wochen nach Akuterkrankung unterscheidet.Eine verzögerte Rekonvaleszenz oder persistierende Beschwerden scheinen nach COVID-19 häufiger als nach anderen akuten Infektionskrankheiten aufzutreten und beinhalten vor allem ein Erschöpfungssyndrom mit Belastungsintoleranz, Brustschmerzen/Belastungsdyspnoe und verschiedene neurologische bzw. neuropsychiatrische Symptome. Diagnostik und Therapie Diagnostik und Therapie sollten interdisziplinär erfolgen. Physiotherapie, psychologische/psychosomatische Anbindung und Möglichkeiten der Rehabilitation sollten erwogen werden. Insbesondere bei persistierender pulmonaler Symptomatik können Patienten von einer Rehabilitationsmaßnahme profitieren. Daten zur Effektivität einer Impfung im Sinne eine Beschwerdelinderung bei Long- COVID fehlen.

Increasing evidence suggests that some patients suffer from persistent symptoms for months after recovery from acute COVID-19. However, the clinical phenotype and its pathogenesis remain unclear. We here present data on complaints and results of a diagnostic workup of patients presenting to the post-COVID clinic at the University Medical Center Freiburg. Retrospective data analysis of persistently symptomatic patients presenting to our clinic at least 6 months after onset of acute COVID-19. All patients were assessed by a doctor and routine laboratory analysis was carried out. Quality of life was assessed using SF-36 questionnaire. In case of specific persisting symptoms, further organ-specific diagnostic evaluation was performed, and patients were referred to respective departments/specialists. 132 Patients (58 male, 74 female; mean age 53.8 years) presented to our clinic at least 6 months after COVID-19. 79 (60 %) had been treated as outpatients and 53 (40 %) as inpatients. Most common complaints were persistent fatigue (82 %) and dyspnea on exertion (61 %). Further common complaints were impairments of concentration (54 %), insomnia (43 %), and impairments of smell or taste (35 %). Quality of life was reduced in all sections of the SF-36 questionnaire, yielding a reduced working capacity. Significant pathological findings in laboratory, echocardiographic and radiological work-up were rare. Impairments in lung function tests were more common in previously hospitalized patients. Patients presenting 6 months after onset of acute COVID-19 suffer from a diverse spectrum of symptoms with impaired quality of life, also referred to as Long COVID or Post-Acute Sequelae of SARS-CoV-2 infection (PASC). Further research is needed to determine the frequency of these post-COVID syndromes and their pathogenesis, natural course and treatment options. Evaluation and management should be multi-disciplinary. Fortbestehende Beschwerden und ärztlich validierte Diagnosen nach COVID-19 sind bislang nicht gut definiert. Wir berichten über Symptome und Diagnostik bei Patienten in unserer COVID-19-Nachsorgeambulanz. Es erfolgte eine retrospektive Analyse der Beschwerden und Befunde bei erwachsenen Patienten nach labordiagnostisch gesicherter COVID-19-Erkrankung mit über mindestens 6 Monate fortbestehenden Beschwerden. Neben Anamnese, körperlicher Untersuchung und Labordiagnostik erfolgte eine symptomgeleitete erweiterte apparative Diagnostik. Die Lebensqualität wurde mittels SF-36-Fragebogens erfasst. 132 Patienten mit über mindestens 6 Monate persistierenden Beschwerden wurden in die Auswertung eingeschlossen (58 männlich, 74 weiblich; mittleres Alter 53,8 Jahre). 40 % der Patienten waren in der Akutphase stationär behandelt worden. Die häufigsten Beschwerden waren Fatigue (82 %), Belastungsdyspnoe (61 %), kognitive Beschwerden (54 %), Schlafstörungen (43 %) und Dysgeusie/-osmie (35 %). Die Lebensqualität war in allen im SF-36 abgefragten Bereichen eingeschränkt. Immerhin 23 % der zuvor Berufstätigen waren aus gesundheitlichen Gründen für mindestens 6 Monate arbeitsunfähig. Signifikante Pathologien in der laborchemischen und apparativen Diagnostik waren insbesondere bei ambulant behandelten Patienten dagegen selten; Lungenfunktionsstörungen waren häufiger bei ehemals stationär behandelten Patienten. Patienten nach COVID-19 berichten über vielfältige Symptome, für die häufig kein organpathologisches Korrelat gefunden werden kann. Dies geht mit Einschränkungen der Lebensqualität und Arbeitsfähigkeit, auch 6 Monate nach Akutinfektion, einher. Populationsbasierte Prävalenz, Pathogenese, Langzeitverlauf und Therapiemöglichkeiten dieses vielfach als Long COVID bezeichneten Syndroms sind weitgehend ungeklärt. Aufgrund der vielfältigen Beschwerden sollte die Patientenversorgung interdisziplinär erfolgen und vermehrt Leistungsdiagnostik und systematische neuropsychiatrische Tests beinhalten.

The SARS-CoV-2 variant of concern (VOC) omicron (B1.1.529) is associated with high infectivity and efficient evasion from humoral immunity induced by previous infection or vaccination. In omicron-infected individuals who have been vaccinated or infected before, severe disease seems to be relatively infrequent pointing towards protection by previously primed SARS-CoV-2-specific T cells that cross-recognize omicron. By performing a comprehensive in-depth comparison of the SARS-CoV-2-specific T cell epitope repertoire after natural infection versus after mRNA vaccination, we here demonstrate that spike-derived epitopes are not dominantly targeted in convalescents compared to non-spike epitopes. In vaccinees, however, we detected a broader spike-specific T cell response compared to convalescents reflected by a more diverse repertoire of dominantly targeted spike-specific T cell epitopes. Booster mRNA vaccination induced a broader spike-specific T cell response in convalescents but not in vaccinees with complete initial vaccination. In convalescents and vaccinees, the targeted T cell epitopes are broadly conserved between ancestral and omicron SARS-CoV-2 variants. Hence, our data emphasize the relevance of mRNA vaccine-induced spike-specific CD8+ T cell responses in combating emerging SARS-CoV-2 VOC including omicron and support the benefit of also boosting convalescent individuals with mRNA vaccines.

Lysine acetylation is a post-translational modification known to regulate protein functions. Here we identify several acetylation sites of the influenza A virus nucleoprotein (NP), including the lysine residues K77, K113 and K229. Viral growth of mutant virus encoding K229R, mimicking a non-acetylated NP lysine residue, is severely impaired compared to wildtype or the mutant viruses encoding K77R or K113R. This attenuation is not the result of decreased polymerase activity, altered protein expression or disordered vRNP co-segregation but rather caused by impaired particle release. Interestingly, release deficiency is also observed mimicking constant acetylation at this site (K229Q), whereas virus encoding NP-K113Q could not be generated. However, mimicking NP hyper-acetylation at K77 and K229 severely diminishes viral polymerase activity, while mimicking NP hypo-acetylation at these sites has no effect on viral replication. These results suggest that NP acetylation at K77, K113 and K229 impacts multiple steps in viral replication of influenza A viruses., Post-translational modifications of influenza A virus proteins can regulate virus replication, but the effect of nucleoprotein (NP) acetylation is not known. Here, Giese et al. identify four NP lysine residues that are acetylated in infected cells and study their role in polymerase activity and virion release.

Scientific Reports 6: Article number: 23138; Published online: 18 March 2016; Updated: 09 May 2016 The Acknowledgements section in this Article is incomplete. “We thank Richard Randall for providing A549 cells stably expressing MxA or shMxA, Georg Kochs for providing the NP-specific antibody and Geoffrey Chase, Georg Kochs and Peter Staeheli for critically reading of the manuscript.

To establish a new lineage in the human population, avian influenza A viruses (AIV) must overcome the intracellular restriction factor MxA. Partial escape from MxA restriction can be achieved when the viral nucleoprotein (NP) acquires the critical human-adaptive amino acid residues 100I/V, 283P, and 313Y. Here, we show that introduction of these three residues into the NP of an avian H5N1 virus renders it genetically unstable, resulting in viruses harboring additional single mutations, including G16D. These substitutions restored genetic stability yet again yielded viruses with varying degrees of attenuation in mammalian and avian cells. Additionally, most of the mutant viruses lost the capacity to escape MxA restriction, with the exception of the G16D virus. We show that MxA escape is linked to attenuation by demonstrating that the three substitutions promoting MxA escape disturbed intracellular trafficking of incoming viral ribonucleoprotein complexes (vRNPs), thereby resulting in impaired nuclear import, and that the additional acquired mutations only partially compensate for this import block. We conclude that for adaptation to the human host, AIV must not only overcome MxA restriction but also an associated block in nuclear vRNP import. This inherent difficulty may partially explain the frequent failure of AIV to become pandemic.

Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A(1-25)) with that of FluB (PB1-B(1-25)) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B(1-25) peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A(1-25) peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.

Phosphorylation at the highly conserved serine residues S23 to S25 in the nuclear export protein (NEP) of influenza A viruses was suspected to regulate its nuclear export activity or polymerase activity-enhancing function. Mutation of these phosphoacceptor sites to either alanine or aspartic acid showed only a minor effect on both activities but revealed the presence of other phosphoacceptor sites that might be involved in regulating NEP activity.

The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase activity in human cells in a concentration-dependent manner. While low NEP levels enhance the polymerase activity, high levels are inhibitory. To gain insights into the underlying mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP resides in the C-terminal moiety and that removal of the last three amino acids completely abrogates this activity. Moreover, compared to full-length NEP, the C-terminal moiety alone exhibited significantly higher activity and seemed to be deregulated, since even the highest concentration did not result in an inhibition of polymerase activity. To determine transient interactions between the N- and C-terminal domains in cis , we fused both ends of NEP to a split click beetle luciferase and performed fragment complementation assays. With decreasing temperature, increased luciferase activity was observed, suggesting that intramolecular binding between the C- and N-terminal domains is preferentially stabilized at low temperatures. This stabilizing effect was significantly reduced with the adaptive mutation M16I or a combination of adaptive mutations (M16I, Y41C, and E75G), which further increased polymerase activity also at 34°C. We therefore propose a model in which the N-terminal moiety of NEP exerts an inhibitory function by back-folding to the C-terminal domain. In this model, adaptive mutations in NEP decrease binding between the C- and N-terminal domains, thereby allowing the protein to “open up” and become active already at a low temperature.

To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/β receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.

To cross the species barrier and establish a new lineage in humans avian influenza A viruses (FLUAV) have to overcome innate immune responses of the host. The IFN-induced large dynamin-like GTPase MxA is a key component in antiviral defense against FLUAV. Human-adapted FLUAV carry an amino acid motive in their nucleoprotein that confers MxA-resistance. This motive derived from the pandemic 1918 virus consists of three amino acids (100V, 283P, 313Y) and is highly conserved in human circulating strains but is absent in all known avian strains. Moreover, this motive never emerged in patients infected with highly pathogenic H5N1 viruses. The latter finding suggests that MxA-resistance might be difficult to achieve for H5N1 viruses. We can now show that human-derived H5N1 viruses, artificially harboring the three MxA-resistance conferring amino acids in their NPs exhibit impaired viral growth. This can be observed in all cell culture systems tested, including cell lines of mammalian and avian origin. Dissecting individual steps in the viral life cycle revealed that this is due to diminished nuclear import of incoming vRNPs. This recombinant virus is genetically instable and acquired single compensatory mutations in NP (D51N, G16D, D101Y, Y385C). With exception of G16D, these compensatory mutations abrogated MxA-resistance. However, these compensatory mutations are not able to completely restore efficient nuclear vRNP import and thus viral fitness. We therefore speculate that gaining MxA-resistance is at the expense of viral fitness – a hurdle that might be too high for H5N1 viruses to establish a new lineage in the human population.