Safety and efficacy of the mRNA BNT162b2 vaccine against SARS-CoV-2 in five groups of immunocompromised patients and healthy controls in a prospective open-label clinical trial

BackgroundPatients with immunocompromised disorders have mainly been excluded from clinical trials of vaccination against COVID-19. Thus, the aim of this prospective clinical trial was to investigate the safety and efficacy after two doses of BNT162b2 mRNA vaccination in five selected groups of immunocompromised patients and healthy controls. Methods539 study subjects (449 patients and 90 controls) were included in the clinical trial. The patients had either primary (n=90), or secondary immunodeficiency disorders due to human immunodeficiency virus infection (n=90), allogeneic hematopoietic stem cell transplantation/chimeric antigen receptor T cell therapy (n=90), solid organ transplantation (SOT) (n=89), or chronic lymphocytic leukemia (CLL) (n=90). The primary endpoint was seroconversion rate two weeks after the second dose. The secondary endpoints were safety and documented SARS-CoV-2 infection. FindingsAdverse events were generally mild, but one case of fatal suspected unexpected serious adverse reaction occurred. 72{middle dot}2% of the immunocompromised patients seroconverted compared to 100% of the controls (p=0.004). Lowest seroconversion rates were found in the SOT (43{middle dot}4%) and CLL (63{middle dot}3%) patient groups with observed negative impact of treatment with mycophenolate mofetil and ibrutinib, respectively. InterpretationThe results showed that the mRNA BNT162b2 vaccine was safe in immunocompromised patients. The rate of seroconversion was substantially lower than in healthy controls, with a wide range of rates and antibody titres among predefined patient groups and subgroups. This clinical trial highlights the need for additional vaccine doses in certain immunocompromised patient groups and/or subgroups to improve immunity. FundingKnut and Alice Wallenberg Foundation, Nordstjernan AB, Region Stockholm, Swedish Research Council, Karolinska Institutet, and organizations for PID/CLL-patients in Sweden.

Longitudinal characterization of humoral and cellular immunity in hospitalized COVID-19 patients reveal immune persistence up to 9 months after infection

BackgroundInsights into early, specific humoral and cellular responses to infection with SARS-CoV-2, as well as the persistence and magnitude of resulting immune memory is important amidst the ongoing pandemic. The combination of humoral and cellular immunity will most likely contribute to protection from reinfection or severe disease. MethodsHere, we conducted a longitudinal study on hospitalized moderate and severe COVID-19 patients from the acute phase of disease into convalescence at five- and nine-months post symptom onset. Utilizing flow cytometry, serological assays as well as B cell and T cell FluoroSpot assays, we assessed the magnitude and specificity of humoral and cellular immune memory during and after human SARS-CoV-2 infection. FindingsDuring acute COVID-19, we observed an increase in germinal center activity, a substantial expansion of antibodysecreting cells, and the generation of SARS-CoV-2-neutralizing antibodies. Despite gradually decreasing antibody levels, we show persistent, neutralizing antibody titers as well as robust specific memory B cell responses and polyfunctional T cell responses at five- and nine-months after symptom onset in both moderate and severe COVID-19 patients. Long-term SARS-CoV-2 specific responses were marked by preferential targeting of spike over nucleocapsid protein. ConclusionsOur findings describe the initiation and, importantly, persistence of cellular and humoral SARS-CoV-2 specific immunological memory in hospitalized COVID-19 patients long after recovery, likely contributing towards protection against reinfection.

Persisting Salivary IgG against SARS-CoV-2 at 9 Months After Mild COVID-19: A Complementary Approach to Population Surveys

BackgroundDeclining humoral immunity in COVID-19 patients and possibility of reinfections has raised concern. Mucosal immunity particularly salivary antibodies could be short-lived. However, long-term studies are sparse. MethodsUsing a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (n=74, Cohort 1), undiagnosed individuals with self-reported questionnaires (n=147, Cohort 2), and individuals sampled pre-pandemic time (n= 35, Cohort 3). ResultsSalivary IgG antibody responses in Cohort 1 (mainly mild COVID-19) were detectable up to 9 month recovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in saliva in majority as seen in blood serology. Salivary IgA was rarely detected at this timepoint. In Cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19 like symptoms. Salivary IgG also tolerated temperature and detergent pre-treatments. ConclusionsUnlike SARS-CoV-2 salivary IgA that appeared short-lived, the specific IgG in saliva appears stable even after mild COVID-19 as noted for blood serology. The non-invasive saliva-based SARS-Cov-2 antibody testing with self-collection at homes may thus serve as a complementary alternative to conventional blood serology.

High-dimensional profiling reveals phenotypic heterogeneity and disease-specific alterations of granulocytes in COVID-19

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in SARS-CoV-2-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion and migration of granulocytes (e.g. CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers supporting pathophysiologic relevance. Furthermore, clinical features, including multi-organ dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19. SignificanceAccumulating evidence shows that granulocytes are key modulators of the immune response to SARS-CoV-2 infection and their dysregulation could significantly impact COVID-19 severity and patient recovery after virus clearance. In the present study, we identify selected immune traits in neutrophil, eosinophil and basophil subsets associated to severity of COVID-19 and to peripheral protein profiles. Moreover, computational modeling indicates that the combined use of phenotypic data and laboratory measurements can effectively predict key clinical outcomes in COVID-19 patients. Finally, patient-matched longitudinal analysis shows phenotypic normalization of granulocyte subsets 4 months after hospitalization. Overall, in this work we extend the current understanding of the distinct contribution of granulocyte subsets to COVID-19 pathogenesis.

Innate lymphoid cell composition associates with COVID-19 disease severity

ObjectivesThe role of innate lymphoid cells (ILCs) in coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is unknown. Understanding the immune response in COVID-19 could contribute to unravel the pathogenesis and identification of treatment targets. To describe the phenotypic landscape of circulating ILCs in COVID-19 patients and to identify ILC phenotypes correlated to serum biomarkers, clinical markers, and laboratory parameters relevant in COVID-19. MethodsBlood samples collected from moderately (n=11) and severely ill (n=12) COVID-19 patients as well as healthy control donors (n=16), were analyzed with 18-parameter flow cytometry. Using supervised and unsupervised approaches, we examined the ILC activation status and homing profile. Clinical and laboratory parameters were obtained from all COVID-19 patients and serum biomarkers were analyzed with multiplex immunoassays. ResultsILCs were largely depleted from the circulation of COVID-19 patients compared with healthy controls. Remaining circulating ILCs from patients revealed increased frequencies of ILC2 in moderate COVID-19, with a concomitant decrease of ILC precursors (ILCp), as compared with controls. ILC2 and ILCp showed an activated phenotype with increased CD69 expression, whereas expression levels of the chemokine receptors CXCR3 and CCR4 were significantly altered in ILC2 and ILCp, and ILC1, respectively. The activated ILC profile of COVID-19 patients was associated with soluble inflammatory markers, while frequencies of ILC subsets were correlated with laboratory parameters that reflect the disease severity. ConclusionThis study provides insights into the potential role of ILCs in immune responses against SARS-CoV-2, particularly linked to the severity of COVID-19.

MAIT cell activation and dynamics associated with COVID-19 disease severity and outcome

Severe COVID-19 is characterized by excessive inflammation of the lower airways. The balance of protective versus pathological immune responses in COVID-19 is incompletely understood. Mucosa-associated invariant T (MAIT) cells are antimicrobial T cells that recognize bacterial metabolites, and can also function as innate-like sensors and mediators of antiviral responses. Here, we investigated the MAIT cell compartment in COVID-19 patients with moderate and severe disease, as well as in convalescence. We show profound and preferential decline in MAIT cells in circulation of patients with active disease paired with strong activation, as well as significant MAIT cell enrichment and pro-inflammatory IL-17A bias in the airways. Unsupervised analysis identified MAIT cell CD69high and CXCR3low immunotypes associated with poor clinical outcome. MAIT cell levels normalized in the convalescent phase, consistent with dynamic recruitment to the tissues and subsequent release with disease resolution. These findings indicate that MAIT cells are engaged in the immune response against SARS-CoV-2 and suggest their involvement in COVID-19 immunopathogenesis. One sentence summaryMAIT cells are strongly activated by SARS-CoV-2 infection in a manner associated with disease severity and outcome, they decline in blood, are enriched in the airways as a prominent IL-17A expressing subset, and dynamically recover in circulation during convalescence.

Perturbations in the mononuclear phagocyte landscape associated with COVID-19 disease severity

Monocytes and dendritic cells are crucial mediators of innate and adaptive immune responses during viral infection, but misdirected responses by these cells might contribute to immunopathology. A comprehensive map of the mononuclear phagocyte (MNP) landscape during SARS-CoV-2 infection and concomitant COVID-19 disease is lacking. We performed 25-color flow cytometry-analysis focusing on MNP lineages in SARS-CoV-2 infected patients with moderate and severe COVID-19. While redistribution of monocytes towards intermediate subset and decrease in circulating DCs occurred in response to infection, severe disease associated with appearance of Mo-MDSC-like cells and a higher frequency of pre-DC2. Furthermore, phenotypic alterations in MNPs, and their late precursors, were cell-lineage specific and in select cases associated with severe disease. Finally, unsupervised analysis revealed that the MNP profile, alone, could identify a cluster of COVID-19 non-survivors. This study provides a reference for the MNP response to clinical SARS-CoV-2 infection and unravel myeloid dysregulation associated with severe COVID-19.

Disease-associated antibody phenotypes and probabilistic seroprevalence estimates during the emergence of SARS-CoV-2

Serological studies are critical for understanding pathogen-specific immune responses and informing public health measures1,2. Here, we evaluate tandem IgM, IgG and IgA responses in a cohort of individuals PCR+ for SARS-CoV-2 RNA (n=105) representing different categories of disease severity, including mild and asymptomatic infections. All PCR+ individuals surveyed were IgG-positive against the virus spike (S) glycoprotein. Elevated Ab levels were associated with hospitalization, with IgA titers, increased circulating IL-6 and strong neutralizing responses indicative of intensive care status. Additional studies of healthy blood donors (n=1,000) and pregnant women (n=900), sampled weekly during the initial outbreak in Stockholm, Sweden (weeks 14-25, 2020), demonstrated that anti-viral IgG titers differed over 1,000-fold between seroconverters, highlighting the need for careful evaluation of assay cut-offs for individual measurements and accurate estimates of seroprevalence (SP). To provide a solution to this, we developed probabilistic machine learning approaches to assign likelihood of past infection without setting an assay cut-off, allowing for more quantitative individual and population-level Ab measures. Using these tools, that considered responses against both S and RBD, we report SARS-CoV-2 S-specific IgG in 6.8% of blood donors and pregnant women two months after the peak of spring COVID-19 deaths, with the SP curve and country death rate following similar trajectories.

Natural killer cell activation related to clinical outcome of COVID-19

Understanding innate immune responses in COVID-19 is important for deciphering mechanisms of host responses and interpreting disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections, but might also contribute to immune pathology. Here, using 28-color flow cytometry, we describe a state of strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients, a pattern mirrored in scRNA-seq signatures of lung NK cells. Unsupervised high-dimensional analysis identified distinct immunophenotypes that were linked to disease severity. Hallmarks of these immunophenotypes were high expression of perforin, NKG2C, and Ksp37, reflecting a high presence of adaptive NK cell expansions in circulation of patients with severe disease. Finally, arming of CD56bright NK cells was observed in course of COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This provides a detailed map of the NK cell activation-landscape in COVID-19 disease.

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

ABSTRACTSARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. We systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in a large cohort of unexposed individuals as well as exposed family members and individuals with acute or convalescent COVID-19. Acute phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative family members and individuals with a history of asymptomatic or mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits robust memory T cell responses akin to those observed in the context of successful vaccines, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19 also in seronegative individuals.Competing Interest StatementThe authors have declared no competing interest.View Full Text