Risk factors for impaired respiratory function post COVID-19: A prospective cohort study of nonhospitalized and hospitalized patients.

BACKGROUND: Severe COVID-19 increases the risk for long-term respiratory impairment, but data after mild COVID-19 are scarce. Our aims were to determine risk factors for reduced respiratory function 3-6 months after COVID-19 infection and to investigate if reduced respiratory function would relate to impairment of exercise performance and breathlessness. METHODS: Patients with COVID-19 were enrolled at the University Hospitals of Umeå and Örebro, and Karlstad Central Hospital, Sweden. Disease severity was defined as mild (nonhospitalized), moderate (hospitalized with or without oxygen treatment), and severe (intensive care). Spirometry, including diffusion capacity (DLCO ), was performed 3-6 months after hospital discharge or study enrollment (for nonhospitalized patients). Breathlessness (defined as ≥1 according to the modified Medical Research Council scale) and functional exercise capacity (1-min sit-to-stand test; 1-MSTST) were assessed. RESULTS: Between April 2020 and May 2021, 337 patients were enrolled in the study. Forced vital capacity and DLCO were significantly lower in patients with severe COVID-19. Among hospitalized patients, 20% had reduced DLCO , versus 4% in nonhospitalized. Breathlessness was found in 40.6% of the participants and was associated with impaired DLCO . A pathological desaturation or heart rate response was observed in 17% of participants during the 1-MSTST. However, this response was not associated with reduced DLCO . CONCLUSION: Reduced DLCO was the major respiratory impairment 3-6 months following COVID-19, with hospitalization as the most important risk factor. The lack of association between impaired DLCO and pathological physiological responses to exertion suggests that these physiological responses are not primarily related to decreased lung function.

Longitudinal dynamics of the human B cell response to the yellow fever 17D vaccine.

A comprehensive understanding of the development and evolution of human B cell responses induced by pathogen exposure will facilitate the design of next-generation vaccines. Here, we utilized a high-throughput single B cell cloning technology to longitudinally track the human B cell response to the yellow fever virus 17D (YFV-17D) vaccine. The early memory B cell (MBC) response was mediated by both classical immunoglobulin M (IgM) (IgM+CD27+) and switched immunoglobulin (swIg+) MBC populations; however, classical IgM MBCs waned rapidly, whereas swIg+ and atypical IgM+ and IgD+ MBCs were stable over time. Affinity maturation continued for 6 to 9 mo following vaccination, providing evidence for the persistence of germinal center activity long after the period of active viral replication in peripheral blood. Finally, a substantial fraction of the neutralizing antibody response was mediated by public clones that recognize a fusion loop-proximal antigenic site within domain II of the viral envelope glycoprotein. Overall, our findings provide a framework for understanding the dynamics and complexity of human B cell responses elicited by infection and vaccination.

At-home sampling to meet geographical challenges for serological assessment of SARS-CoV-2 exposure in a rural region of northern Sweden, March to May 2021: a retrospective cohort study.

BackgroundThe current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture for testing is usually performed by trained staff at healthcare centres. Long travel distances to healthcare centres in rural regions may introduce a bias of testing towards relatively large communities with closer access. Rural regions are therefore often not represented in population-based data.AimThe aim of this retrospective cohort study was to develop and implement a strategy for at-home testing in a rural region of Sweden during spring 2021, and to evaluate its role to provide equal health care for its inhabitants.MethodsWe developed a sensitive method to measure antibodies to the S-protein of SARS-CoV-2 and optimised this assay for clinical use together with a strategy of at-home capillary blood sampling.ResultsWe demonstrated that our ELISA gave comparable results after analysis of capillary blood or serum from SARS-CoV-2-experienced individuals. We demonstrated stability of the assay under conditions that reflected temperature and humidity during winter or summer. By assessment of capillary blood samples from 4,122 individuals, we could show both feasibility of the strategy and that implementation shifted the geographical spread of testing in favour of rural areas.ConclusionImplementation of at-home sampling enabled citizens living in remote rural areas access to centralised and sensitive laboratory antibody tests. The strategy for testing used here could therefore enable disease control authorities to get rapid access to information concerning immunity to infectious diseases, even across vast geographical distance.

Sterile inflammation in the spleen during atherosclerosis provides oxidation-specific epitopes that induce a protective B-cell response.

The B-cell response in atherosclerosis is directed toward oxidation-specific epitopes such as phosphorylcholine (PC) that arise during disease-driven oxidation of self-antigens. PC-bearing antigens have been used to induce atheroprotective antibodies against modified low-density lipoproteins (oxLDL), leading to plaque reduction. Previous studies have found that B-cell transfer from aged atherosclerotic mice confers protection to young mice, but the mechanism is unknown. Here, we dissected the atheroprotective response in the spleen and found an ongoing germinal center reaction, accumulation of antibody-forming cells, and inflammasome activation in apolipoprotein E-deficient mice (Apoe(-/-)). Specific B-cell clone expansion involved the heavy chain variable region (Vh) 5 and Vh7 B-cell receptor families that harbor anti-PC reactivity. oxLDL also accumulated in the spleen. To investigate whether protection could be induced by self-antigens alone, we injected apoptotic cells that carry the same oxidation-specific epitopes as oxLDL. This treatment reduced serum cholesterol and inhibited the development of atherosclerosis in a B-cell-dependent manner. Thus, we conclude that the spleen harbors a protective B-cell response that is initiated in atherosclerosis through sterile inflammation. These data highlight the importance of the spleen in atherosclerosis-associated immunity.

Independent expansion of epitope-specific plasma cell responses upon HIV-1 envelope glycoprotein immunization.

Abs that bind the functional envelope glycoprotein (Env) spike are considered critical for a broadly effective prophylactic HIV-1 vaccine. The difficulty in eliciting such Abs by vaccination is partially attributed to the immunodominance of hydrophilic, surface-exposed variable protein regions of Env. However, little is known about the potential for competition between B cells that recognize distinct and distal epitopes on Env during protein subunit vaccination. In this study, we address this basic question at the level of Ab-secreting cells and serum IgG using a pair of isogenic soluble Env trimers, designated wildtype and gV3, which differ only in their potential to activate B cell responses against the highly immunogenic V3 region of Env. Immunization of mice with gV3 resulted in a markedly lower Ag-specific response compared with that induced by wildtype Env and could be explained by a loss of V3-directed reactivities. There was no redistribution of the response to other regions of Env in gV3-inoculated mice, suggesting that the epitope-specific Ab-secreting cell responses measured after boost are independently regulated rather than dictated by direct or indirect competition between B cells recognizing different structural elements of Env. This information is relevant for ongoing efforts in Env immunogen design to focus responses on conserved neutralizing determinants and for our general understanding of B cell responses to large-protein Ags that display numerous B cell epitopes.

Human plasmacytoid dendritic cells efficiently capture HIV-1 envelope glycoproteins via CD4 for antigen presentation.

Advances in HIV-1 vaccine clinical trials and preclinical research indicate that the virus envelope glycoproteins (Env) are likely to be an essential component of a prophylactic vaccine. Efficient Ag uptake and presentation by dendritic cells (DCs) is important for strong CD4(+) Th cell responses and the development of effective humoral immune responses. In this study, we examined the capacity of distinct primary human DC subsets to internalize and present recombinant Env to CD4(+) T cells. Consistent with their specific receptor expression, skin DCs bound and internalized Env via C-type lectin receptors, whereas blood DC subsets, including CD1c(+) myeloid DCs, CD123(+) plasmacytoid DCs (PDCs), and CD141(+) DCs exhibited a restricted repertoire of C-type lectin receptors and relied on CD4 for uptake of Env. Despite a generally poor capacity for Ag uptake compared with myeloid DCs, the high expression of CD4 on PDCs allowed them to bind and internalize Env very efficiently. CD4-mediated uptake delivered Env to EEA1(+) endosomes that progressed to Lamp1(+) and MHC class II(+) lysosomes where internalized Env was degraded rapidly. Finally, all three blood DC subsets were able to internalize an Env-CMV pp65 fusion protein via CD4 and stimulate pp65-specific CD4(+) T cells. Thus, in the in vitro systems described in this paper, CD4-mediated uptake of Env is a functional pathway leading to Ag presentation, and this may therefore be a mechanism used by blood DCs, including PDCs, for generating immune responses to Env-based vaccines.

Immunization of macaques with soluble HIV type 1 and influenza virus envelope glycoproteins results in a similarly rapid contraction of peripheral B-cell responses after boosting.

The envelope glycoproteins (Env) represent a critical component of a successful antibody-mediated human immunodeficiency virus type 1 (HIV-1) vaccine. However, immunization with soluble Env was reported to induce short-lived antibody responses, suggesting that Env has unusual immunogenic properties. Here, we directly compared the magnitude and durability of B-cell responses induced by HIV-1 Env and an unrelated soluble viral protein, influenza virus hemagglutinin (HA), in simultaneously inoculated macaques. We demonstrate robust peak responses followed by rapid contraction of circulating antibody and memory B cells for both antigens, suggesting that short-lived responses are not unique to HIV-1 Env but may be a common feature of soluble protein vaccines.

Intravenous immunoglobulin therapy for COVID-19 in immunocompromised patients: A retrospective cohort study.

OBJECTIVES: To investigate the effectiveness of intravenous immunoglobulin (IVIG) as treatment for COVID-19 in immunocompromised patients. METHODS: This retrospective study investigated outcomes for immunocompromised, vaccine non-responsive, patients that between September 2022 and April 2023 received IVIG as treatment for COVID-19 in the region of Västerbotten, Sweden. We analyzed clinical data, viral load, and anti-SARS-CoV-2 IgG binding and neutralization levels of patient serum samples and IVIG production batches. Primary and secondary outcomes were clinical cure and viral clearance, respectively. RESULTS: Sixteen patients were analyzed. After a median COVID-19 duration of 4 weeks, a median 60 g IVIG infusion increased SARS-CoV-2 binding and neutralizing antibody levels, with broad in vitro activity against tested variants. The treatment resulted in abrogation of viremia in all patients and general improvement in 15 survivors that all met the primary endpoint. Thirteen patients met the secondary endpoint at follow-up after a median of four months. Two subjects with persistent SARS-CoV-2 carriage relapsed but were successfully retreated with IVIG. CONCLUSIONS: Antibodies in IVIG efficiently neutralized several SARS-CoV-2 variants. Treatment with IVIG was associated with clinical cure and viral clearance in immunocompromised patients. Our data suggests that IVIG could be a novel treatment alternative for COVID-19 for this patient category.

Olfactory dysfunction as an early predictor for post-COVID condition at 1-year follow-up.

BACKGROUND: Olfactory dysfunction together with neurological and cognitive symptoms are common after COVID-19. We aimed to study whether performance on olfactory and neuropsychological tests following infection predict post-COVID condition (PCC), persisting symptoms, and reduced health-related quality of life. METHODS: Both hospitalized (N = 10) and non-hospitalized individuals (N = 56) were enrolled in this prospective cohort study. Participants were evaluated 1-3 months after infection with an olfactory threshold test and neuropsychological tests, which was used as predictors of PCC. A questionnaire outlining persisting symptoms and the validated instrument EuroQol five-dimension five-level for health-related quality of life assessment were used as outcome data 1 year after infection (N = 59). Principal component analysis was used to identify relevant predictors for PCC at 1 year. RESULTS: Objectively assessed olfactory dysfunction at 1-3 months post infection, but not subjective olfactory symptoms, predicted post-COVID condition with reduced health-related quality of life (PCC+) at 1 year. The PCC+ group scored more often below the cut off for mild cognitive impairment on the Montreal Cognitive Assessment (61.5% vs. 21.7%) and higher on the Multidimensional Fatigue Inventory-20, compared to the group without PCC+. CONCLUSION: Our results indicate that objectively assessed, olfactory dysfunction is a predictor for PCC+. These findings underscore the importance of objective olfactory testing. We propose that olfactory screening in the early post-acute phase of COVID-19 infection might identify individuals that are at higher risk of developing long-term health sequalae.

Direct and indirect effects of Puumala hantavirus on platelet function.

Thrombocytopenia is a cardinal symptom of hantavirus-induced diseases including Puumala virus (PUUV)-induced hemorrhagic fever with renal syndrome (HFRS), which is associated with impaired platelet function, bleeding manifestations and augmented thrombotic risk. However, the underlying mechanisms causing thrombocytopenia and platelet hypo-responsiveness are unknown. Thus, we investigated the direct and indirect impact of PUUV on platelet production, function and degradation. Analysis of PUUV-HFRS patient blood revealed that platelet hypo-responsiveness in PUUV infection was cell-intrinsic and accompanied by reduced platelet-leukocyte aggregates (PLAs) and upregulation of monocyte tissue factor (TF), whereas platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation was comparable to healthy controls. Plasma CXCL4 levels followed platelet count dynamics throughout disease course. PUUV activated both neutrophils and monocytes in vitro, but platelet desialylation, degranulation and GPIIb/IIIa activation as well as PLA formation and endothelial adhesion under flow remained unaltered in the presence of PUUV. Further, MEG-01 megakaryocytes infected with PUUV displayed unaltered polyploidization, expression of surface receptors and platelet production. However, infection of endothelial cells with PUUV significantly increased platelet sequestration. Our data thus demonstrate that although platelet production, activation or degradation are not directly modulated, PUUV indirectly fosters thrombocytopenia by sequestration of platelets to infected endothelium. Upregulation of immunothrombotic processes in PUUV-HFRS may further contribute to platelet dysfunction and consumption. Given the pathophysiologic similarities of hantavirus infections, our findings thus provide important insights into the mechanisms underlying thrombocytopenia and highlight immune-mediated coagulopathy as potential therapeutic target.

Semimethylation is a feature of diffuse large B-cell lymphoma, and subgroups with poor prognosis are characterized by global hypomethylation and short telomere length.

BACKGROUND: Large B-cell lymphoma (LBCL) is the most common lymphoma and is known to be a biologically heterogeneous disease regarding genetic, phenotypic, and clinical features. Although the prognosis is good, one-third has a primary refractory or relapsing disease which underscores the importance of developing predictive biological markers capable of identifying high- and low-risk patients. DNA methylation (DNAm) and telomere maintenance alterations are hallmarks of cancer and aging. Both these alterations may contribute to the heterogeneity of the disease, and potentially influence the prognosis of LBCL. RESULTS: We studied the DNAm profiles (Infinium MethylationEPIC BeadChip) and relative telomere lengths (RTL) with qPCR of 93 LBCL cases: Diffuse large B-cell lymphoma not otherwise specified (DLBCL, n = 66), High-grade B-cell lymphoma (n = 7), Primary CNS lymphoma (n = 8), and transformation of indolent B-cell lymphoma (n = 12). There was a substantial methylation heterogeneity in DLBCL and other LBCL entities compared to normal cells and other B-cell neoplasms. LBCL cases had a particularly aberrant semimethylated pattern (0.15 ≤ ß ≤ 0.8) with large intertumor variation and overall low hypermethylation (ß > 0.8). DNAm patterns could not be used to distinguish between germinal center B-cell-like (GC) and non-GC DLBCL cases. In cases treated with R-CHOP-like regimens, a high percentage of global hypomethylation (ß < 0.15) was in multivariable analysis associated with worse disease-specific survival (DSS) (HR 6.920, 95% CI 1.499-31.943) and progression-free survival (PFS) (HR 4.923, 95% CI 1.286-18.849) in DLBCL and with worse DSS (HR 5.147, 95% CI 1.239-21.388) in LBCL. These cases with a high percentage of global hypomethylation also had a higher degree of CpG island methylation, including islands in promoter-associated regions, than the cases with less hypomethylation. Additionally, telomere length was heterogenous in LBCL, with a subset of the DLBCL-GC cases accounting for the longest RTL. Short RTL was independently associated with worse DSS (HR 6.011, 95% CI 1.319-27.397) and PFS (HR 4.689, 95% CI 1.102-19.963) in LBCL treated with R-CHOP-like regimens. CONCLUSION: We hypothesize that subclones with high global hypomethylation and hypermethylated CpG islands could have advantages in tumor progression, e.g. by inactivating tumor suppressor genes or promoting treatment resistance. Our findings suggest that cases with high global hypomethylation and thus poor prognosis could be candidates for alternative treatment regimens including hypomethylating drugs.

Hybrid capture-based next-generation sequencing of new and old world Orthohantavirus strains and wild-type Puumala isolates from humans and bank voles.

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5-40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced. We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein. Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes.

Modulation of innate immune response to mRNA vaccination after SARS-CoV-2 infection or sequential vaccination in humans.

mRNA vaccines are likely to become widely used for the prevention of infectious diseases in the future. Nevertheless, a notable gap exists in mechanistic data, particularly concerning the potential effects of sequential mRNA immunization or preexisting immunity on the early innate immune response triggered by vaccination. In this study, healthy adults, with or without documented prior SARS-CoV-2 infection, were vaccinated with the BNT162b2/Comirnaty mRNA vaccine. Prior infection conferred significantly stronger induction of proinflammatory and type I IFN-related gene signatures, serum cytokines, and monocyte expansion after the prime vaccination. The response to the second vaccination further increased the magnitude of the early innate response in both study groups. The third vaccination did not further increase vaccine-induced inflammation. In vitro stimulation of PBMCs with TLR ligands showed no difference in cytokine responses between groups, or before or after prime vaccination, indicating absence of a trained immunity effect. We observed that levels of preexisting antigen-specific CD4 T cells, antibody, and memory B cells correlated with elements of the early innate response to the first vaccination. Our data thereby indicate that preexisting memory formed by infection may augment the innate immune activation induced by mRNA vaccines.

SARS-CoV-2 infection induces hyaluronan production in vitro and hyaluronan levels in COVID-19 patients relate to morbidity and long-term lung impairment: a prospective cohort study.

We previously demonstrated that the lungs of deceased COVID-19 patients were filled with a clear hydrogel consisting of hyaluronan (HA). In this translational study, we investigated the role of HA at all stages of COVID-19 disease to map the consequences of elevated HA on morbidity and identify the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced HA production. A reduced alveolar surface area was observed in the lungs of deceased COVID-19 patients compared to healthy controls, as visualized by a 3D rendering of lung morphology using light-sheet fluorescence microscopy. We confirmed the presence of HA in lung biopsies and found large quantities of proinflammatory fragmented HA. The association of systemic HA in blood plasma and disease severity was assessed in patients with mild (WHO Clinical Progression Scale, WHO-CPS, 1-5) and severe COVID-19 (WHO-CPS, 6-9) during the acute and convalescent phases and related to lung function. We found that systemic levels of HA were high during acute COVID-19 disease, remained elevated during convalescence, and were associated with a reduced diffusion capacity. In vitro 3D-lung models, differentiated from primary human bronchial epithelial cells, were used to study the effects of SARS-CoV-2 infection on HA metabolism, and transcriptomic analyses revealed a dysregulation of HA synthases and hyaluronidases, both contributing to increased HA in apical secretions. Furthermore, corticosteroid treatment reduced the inflammation and downregulated HA synthases. Our findings demonstrate that HA plays a role in COVID-19 morbidity and that sustained elevated HA concentrations may contribute to long-term respiratory impairment.IMPORTANCEThis study provides insights into the role of hyaluronan (HA) in the severity and long-term impact of COVID-19 on lung function. Through extensive morphological examination of lung tissues and a multicenter study, we identified that HA levels are significantly elevated in COVID-19 patients, correlating with a reduced lung diffusion capacity during convalescence. Using a 3D-lung model, we further uncovered how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 infection causes a dysregulated HA metabolism, leading to increased HA production. Our findings provide valuable insights into the pathogenesis of SARS-CoV-2 and suggest that targeting HA metabolism could offer new therapeutic avenues for managing COVID-19, particularly to prevent long-term lung impairment. Additionally, HA holds potential as a biomarker for predicting disease severity, which could guide personalized treatment strategies.

Biochemically defined HIV-1 envelope glycoprotein variant immunogens display differential binding and neutralizing specificities to the CD4-binding site.

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.

Immune response to SARS-CoV-2 mRNA vaccination in multiple sclerosis patients after rituximab treatment interruption.

Peripheral B cell depletion via anti-CD20 treatment is a highly effective disease-modifying treatment for reducing new relapses in multiple sclerosis (MS) patients. A drawback of rituximab (RTX) and other anti-CD20 antibodies is a poor immune response to vaccination. While this can be mitigated by treatment interruption of at least six months prior to vaccination, the timing to resume treatment while maintaining subsequent vaccine responses remains undetermined. Here, we characterized SARS-CoV-2 S-directed antibody and B cell responses throughout three BNT162b2 mRNA vaccine doses in RTX-treated MS patients, with the first two doses given during treatment interruption. We examined B-cell mediated immune responses in blood samples from patients with RTX-treated MS throughout three BNT162b2 vaccine doses, compared to an age- and sex-matched healthy control group. The first vaccine dose was given 1.3 years (median) after the last RTX infusion, the second dose one month after the first, and the third dose four weeks after treatment re-initiation. We analyzed SARS-CoV-2 S-directed antibody levels using enzyme-linked immunosorbent assay (ELISA), and the neutralization capacity of patient serum against SARS-CoV-2 S-pseudotyped lentivirus using luciferase reporter assay. In addition, we assessed switched memory (CD19+CD20+CD27+IgD-), unswitched memory (CD19+CD20+CD27+IgD+), naïve (CD19+CD20+CD27-IgD+), and double negative (DN, CD19+CD20+CD27-IgD-) B cell frequencies, as well as their SARS-CoV-2 S-specific (CoV+) and Decay Accelerating Factor-negative (DAF-) subpopulations, using flow cytometry. After two vaccine doses, S-binding antibody levels and neutralization capacity in SARS-CoV-2-naïve MS patients were comparable to vaccinated healthy controls, albeit with greater variation. Higher antibody response levels and CoV+-DN B cell frequencies after the second vaccine dose were predictive of a boost effect after the third dose, even after re-initiation of rituximab treatment. MS patients also exhibited lower frequencies of DAF- memory B cells, a suggested proxy for germinal centre activity, than control individuals. S-binding antibody levels in RTX-treated MS patients after two vaccine doses could help determine which individuals would need to move up their next vaccine booster dose or postpone their next RTX infusion. Our findings also offer first indications on the potential importance of antigenic stimulation of DN B cells and long-term impairment of germinal centre activity in rituximab-treated MS patients.

Extracellular Galectin 4 Drives Immune Evasion and Promotes T-cell Apoptosis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell-produced protein that was deposited into the ECM of PDAC tumors and detected high-circulating levels of gal 4 in patients with PDAC. In orthotopic transplantation experiments, we observed increased infiltration of T cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1-/- mice, demonstrating that the effect was mediated by the adaptive immune system. By performing single-cell RNA-sequencing, we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression associated with a higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors with reduced gal 4 expression. Using a coculture system, we observed that extracellular gal 4 induced apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.

High prevalence of persistent symptoms and reduced health-related quality of life 6 months after COVID-19.

Background: The long-term sequelae after COVID-19 constitute a challenge to public health and increased knowledge is needed. We investigated the prevalence of self-reported persistent symptoms and reduced health-related quality of life (HRQoL) in relation to functional exercise capacity, 6 months after infection, and explored risk factors for COVID-19 sequalae. Methods: This was a prospective, multicenter, cohort study including 434 patients. At 6 months, physical exercise capacity was assessed by a 1-minute sit-to-stand test (1MSTST) and persistent symptoms were reported and HRQoL was evaluated through the EuroQol 5-level 5-dimension (EQ-5D-5L) questionnaire. Patients with both persistent symptoms and reduced HRQoL were classified into a new definition of post-acute COVID syndrome, PACS+. Risk factors for developing persistent symptoms, reduced HRQoL and PACS+ were identified by multivariable Poisson regression. Results: Persistent symptoms were experienced by 79% of hospitalized, and 59% of non-hospitalized patients at 6 months. Hospitalized patients had a higher prevalence of self-assessed reduced overall health (28 vs. 12%) and PACS+ (31 vs. 11%). PACS+ was associated with reduced exercise capacity but not with abnormal pulse/desaturation during 1MSTST. Hospitalization was the most important independent risk factor for developing persistent symptoms, reduced overall health and PACS+. Conclusion: Persistent symptoms and reduced HRQoL are common among COVID-19 survivors, but abnormal pulse and peripheral saturation during exercise could not distinguish patients with PACS+. Patients with severe infection requiring hospitalization were more likely to develop PACS+, hence these patients should be prioritized for clinical follow-up after COVID-19.

Vaccination of SARS-CoV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages.

Vaccination of SARS-CoV-2 convalescent individuals generates broad and potent antibody responses. Here, we isolate 459 spike-specific monoclonal antibodies (mAbs) from two individuals who were infected with the index variant of SARS-CoV-2 and later boosted with mRNA-1273. We characterize mAb genetic features by sequence assignments to the donors' personal immunoglobulin genotypes and assess antibody neutralizing activities against index SARS-CoV-2, Beta, Delta, and Omicron variants. The mAbs used a broad range of immunoglobulin heavy chain (IGH) V genes in the response to all sub-determinants of the spike examined, with similar characteristics observed in both donors. IGH repertoire sequencing and B cell lineage tracing at longitudinal time points reveals extensive evolution of SARS-CoV-2 spike-binding antibodies from acute infection until vaccination five months later. These results demonstrate that highly polyclonal repertoires of affinity-matured memory B cells are efficiently recalled by vaccination, providing a basis for the potent antibody responses observed in convalescent persons following vaccination.