Pentameric C-reactive protein is a better prognostic biomarker and remains elevated for longer than monomeric CRP in hospitalized patients with COVID-19.

The differing roles of the pentameric (p) and monomeric (m) C-reactive protein (CRP) isoforms in viral diseases are not fully understood, which was apparent during the COVID-19 pandemic regarding the clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Herein, we investigated the predictive value of the pCRP and mCRP isoforms for COVID-19 severity in hospitalized patients and evaluated how the levels of the protein isoforms changed over time during and after acute illness. This study utilized samples from a well-characterized cohort of Swedish patients with SARS-CoV-2 infection, the majority of whom had known risk factors for severe COVID-19 and required hospitalization. The levels of pCRP were significantly raised in patients with severe COVID-19 and in contrast to mCRP the levels were significantly associated with disease severity. Additionally, the pCRP levels remained elevated for at least six weeks post inclusion, which was longer compared to the two weeks for mCRP. Our data indicates a low level of inflammation lasting for at least six weeks following COVID-19, which might indicate that the disease has an adverse effect on the immune system even after the viral infection is resolved. It is also clear that the current standard method of testing pCRP levels upon hospitalization is a useful marker for predicting disease severity and mCRP testing would not add any clinical relevance for patients with COVID-19.

T cell perturbations persist for at least 6 months following hospitalization for COVID-19.

COVID-19 is being extensively studied, and much remains unknown regarding the long-term consequences of the disease on immune cells. The different arms of the immune system are interlinked, with humoral responses and the production of high-affinity antibodies being largely dependent on T cell immunity. Here, we longitudinally explored the effect COVID-19 has on T cell populations and the virus-specific T cells, as well as neutralizing antibody responses, for 6-7 months following hospitalization. The CD8+ TEMRA and exhausted CD57+ CD8+ T cells were markedly affected with elevated levels that lasted long into convalescence. Further, markers associated with T cell activation were upregulated at inclusion, and in the case of CD69+ CD4+ T cells this lasted all through the study duration. The levels of T cells expressing negative immune checkpoint molecules were increased in COVID-19 patients and sustained for a prolonged duration following recovery. Within 2-3 weeks after symptom onset, all COVID-19 patients developed anti-nucleocapsid IgG and spike-neutralizing IgG as well as SARS-CoV-2-specific T cell responses. In addition, we found alterations in follicular T helper (TFH) cell populations, such as enhanced TFH-TH2 following recovery from COVID-19. Our study revealed significant and long-term alterations in T cell populations and key events associated with COVID-19 pathogenesis.

The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture.

The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies. IMPORTANCE Bunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for humans are not available. The bunyavirus genome is wrapped up by the nucleoprotein (NP) and interacts with the viral polymerase, forming a ribonucleoprotein (RNP). This is the only form of the genome active for viral replication and assembly. However, until now how NPs are organized within an RNP was not known for any orthobunyavirus. Here, we purified RNPs from the prototypical orthobunyavirus, Bunyamwera virus, and employed microscopy approaches to show that the NP portion of the RNP was helical. We then combined our helical average with the known structure of an NP monomer, generating a pseudo-atomic model of this region. This arrangement allowed the RNPs to be highly flexible, which was critical for several stages of the viral replication cycle, such as segment circularization.

Major alterations to monocyte and dendritic cell subsets lasting more than 6 months after hospitalization for COVID-19.

Introduction: After more than two years the Coronavirus disease-19 (COVID-19) pandemic continues to burden healthcare systems and economies worldwide, and it is evident that the effects on the immune system can persist for months post-infection. The activity of myeloid cells such as monocytes and dendritic cells (DC) is essential for correct mobilization of the innate and adaptive responses to a pathogen. Impaired levels and responses of monocytes and DC to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is likely to be a driving force behind the immune dysregulation that characterizes severe COVID-19. Methods: Here, we followed a cohort of COVID-19 patients hospitalized during the early waves of the pandemic for 6-7 months. The levels and phenotypes of circulating monocyte and DC subsets were assessed to determine both the early and long-term effects of the SARS-CoV-2 infection. Results: We found increased monocyte levels that persisted for 6-7 months, mostly attributed to elevated levels of classical monocytes. Myeloid derived suppressor cells were also elevated over this period. While most DC subsets recovered from an initial decrease, we found elevated levels of cDC2/cDC3 at the 6-7 month timepoint. Analysis of functional markers on monocytes and DC revealed sustained reduction in program death ligand 1 (PD-L1) expression but increased CD86 expression across almost all cell types examined. Finally, C-reactive protein (CRP) correlated positively to the levels of intermediate monocytes and negatively to the recovery of DC subsets. Conclusion: By exploring the myeloid compartments, we show here that alterations in the immune landscape remain more than 6 months after severe COVID-19, which could be indicative of ongoing healing and/or persistence of viral antigens.

Soluble Urokinase Plasminogen Activator Receptor (suPAR) Independently Predicts Severity and Length of Hospitalisation in Patients With COVID-19.

Background: Efficient healthcare based on prognostic variables in hospitalised patients with COVID-19 could reduce the risk of complications and death. Recently, soluble urokinase Plasminogen Activator Receptor (suPAR) was shown to predict respiratory failure, kidney injury, and clinical outcome in patients with SARS-CoV-2 infection. The aim of this study was to investigate the value of suPAR as a prognostic tool, in comparison with other variables, regarding disease severity and length of hospital stay in patients with COVID-19. Patients and Methods: Individuals hospitalised with COVID-19 (40 males, 20 females; median age 57.5 years) with a median symptom duration of 10 days and matched, healthy controls (n = 30) were included. Admission levels of suPAR were measured in serum by enzyme-linked immunosorbent assay. Blood cell counts, C-reactive protein (CRP) levels, lactate dehydrogenase (LDH), plasma creatinine and estimated glomerular filtration rates were analysed and oxygen demand, level of care and length of hospitalisation recorded. Results: Patients had significantly higher suPAR levels compared to controls (P < 0.001). Levels were higher in severely/critically (median 6.6 ng/mL) compared with moderately ill patients (median 5.0 ng/mL; P = 0.002). In addition, suPAR levels correlated with length of hospitalisation (rho = 0.35; P = 0.006). Besides suPAR, LDH, CRP, neutrophil count, neutrophil-to-monocyte and neutrophil-to-lymphocyte ratio, body mass index and chronic renal failure were discriminators of COVID-19 severity and/or predictors of length of hospitalisation. Conclusion: Admission levels of suPAR were higher in patients who developed severe/critical COVID-19 and associated with length of hospital stay. In addition, we showed that suPAR functioned as an independent predictor of COVID-19 disease severity.

High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D.

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2ß and CDR3α. This allowed CDR3ß variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.