Serum and Salivary IgG and IgA Response After COVID-19 Messenger RNA Vaccination.

Importance: There is still considerable controversy in the literature regarding the capacity of intramuscular messenger RNA (mRNA) vaccination to induce a mucosal immune response. Objective: To compare serum and salivary IgG and IgA levels among mRNA-vaccinated individuals with or without previous SARS-CoV-2 infection. Design, Setting, and Participants: In this cohort study, SARS-CoV-2-naive participants and those with previous infection were consecutively included in the CoviCompare P and CoviCompare M mRNA vaccination trials and followed up to day 180 after vaccination with either the BNT162b2 (Pfizer-BioNTech) vaccine or the mRNA-1273 (Moderna) vaccine at the beginning of the COVID-19 vaccination campaign (from February 19 to June 8, 2021) in France. Data were analyzed from October 25, 2022, to July 13, 2023. Main Outcomes and Measures: An ultrasensitive digital enzyme-linked immunosorbent assay was used for the comparison of SARS-CoV-2 spike-specific serum and salivary IgG and IgA levels. Spike-specific secretory IgA level was also quantified at selected times. Results: A total of 427 individuals were included in 3 groups: participants with SARS-CoV-2 prior to vaccination who received 1 single dose of BNT162b2 (Pfizer-BioNTech) (n = 120) and SARS-CoV-2-naive individuals who received 2 doses of mRNA-1273 (Moderna) (n = 172) or 2 doses of BNT162b2 (Pfizer-BioNTech) (n = 135). The median age was 68 (IQR, 39-75) years, and 228 (53.4%) were men. SARS-CoV-2 spike-specific IgG saliva levels increased after 1 or 2 vaccine injections in individuals with previous infection and SARS-CoV-2-naive individuals. After vaccination, SARS-CoV-2-specific saliva IgA levels, normalized with respect to total IgA levels, were significantly higher in participants with previous infection, as compared with the most responsive mRNA-1273 (Moderna) recipients (median normalized levels, 155 × 10-5 vs 37 × 10-5 at day 29; 107 × 10-5 vs 54 × 10-5 at day 57; and 104 × 10-5 vs 70 × 10-5 at day 180 [P < .001]). In contrast, compared with day 1, spike-specific IgA levels in the BNT162b2-vaccinated SARS-CoV-2-naive group increased only at day 57 (36 × 10-5 vs 49 × 10-5 [P = .01]). Bona fide multimeric secretory IgA levels were significantly higher in individuals with previous infection compared with SARS-CoV-2-naive individuals after 2 antigenic stimulations (median optical density, 0.36 [IQR, 0.16-0.63] vs 0.16 [IQR, 0.10-0.22]; P < .001). Conclusions and Relevance: The findings of this cohort study suggest that mRNA vaccination was associated with mucosal immunity in individuals without prior SARS-CoV-2 infection, but at much lower levels than in previously infected individuals. Further studies are needed to determine the association between specific saliva IgA levels and prevention of infection or transmission.

Anti-S-layer monoclonal antibodies impact Clostridioides difficile physiology.

Clostridioides difficile (C. difficile), a gram-positive anaerobic and spore-forming bacterium, is the leading cause of nosocomial antibiotic-associated diarrhea in adults which is characterized by high levels of recurrence and mortality. Surface (S)-layer Protein A (SlpA), the most abundantly expressed protein on the bacterial surface, plays a crucial role in the early stages of infection although the nature of its involvement in C. difficile physiology is yet to be fully understood. Anti-S-layer antibodies have been identified in the sera of convalescent patients and have been correlated with improved outcomes of C. difficile infection (CDI). However, the precise mechanisms by which anti-S-layer antibodies confer protection to the host remain unknown. In this study, we report the first monoclonal antibodies (mAbs) targeting the S-layer of reference strain 630. Characterization of these mAbs unraveled important roles for the S-layer protein in growth, toxin secretion, and biofilm formation by C. difficile, with differential and even opposite effects of various anti-SlpA mAbs on these functions. Moreover, one anti-SlpA mAb impaired C. difficile growth and conferred sensitivity to lysozyme-induced lysis. The results of this study show that anti-S-layer antibody responses can be beneficial or harmful for the course of CDI and provide important insights for the development of adequate S-layer-targeting therapeutics.

A monoclonal antibody collection for C. difficile typing ?

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in adults. Various C. difficile strains circulate currently, associated with different outcomes and antibiotic resistance profiles. However, most studies still focus on the reference strain 630 that does not circulate anymore, partly due to the lack of immunological tools to study current clinically important C. difficile PCR ribotypes. The goal of this study was to generate monoclonal antibodies recognizing various epidemic ribotypes of C. difficile. To do so, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various C. difficile strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole bacteria from current C. difficile ribotypes with different cross-specificities. This first collection of anti-C. difficile mAbs represent valuable tools for basic and clinical research.

Identification of natural killer markers associated with fatal outcome in COVID-19 patients.

Introduction: Increasing evidence has shown that coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunological response. Previous studies have demonstrated that natural killer (NK) cell dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of NK cell markers as a driver of death in the most critically ill patients. Methods: We enrolled 50 non-vaccinated hospitalized patients infected with the initial virus or the alpha variant of SARS-CoV-2 with moderate or severe illness, to evaluate phenotypic and functional features of NK cells. Results: Here, we show that, consistent with previous studies, evolution NK cells from COVID-19 patients are more activated, with the decreased activation of natural cytotoxicity receptors and impaired cytotoxicity and IFN-γ production, in association with disease regardless of the SARS-CoV-2 strain. Fatality was observed in 6 of 17 patients with severe disease; NK cells from all of these patients displayed a peculiar phenotype of an activated memory-like phenotype associated with massive TNF-α production. Discussion: These data suggest that fatal COVID-19 infection is driven by an uncoordinated inflammatory response in part mediated by a specific subset of activated NK cells.

Intestinal Candida albicans overgrowth in IgA deficiency.

BACKGROUND: Secretory IgA interacts with commensal bacteria, but its impact on human mycobiota ecology has not been widely explored. In particular, whether human IgA-deficiency is associated with gut fungal dysbiosis remains unknown. OBJECTIVES: Our goal was to study the impact of IgA on gut mycobiota ecology. METHODS: The Fungi-Flow method was used to characterize fecal, systemic, and maternal IgA, IgM, and IgG responses against 14 representative fungal strains (yeast/spores or hyphae forms) in healthy donors (HDs) (n = 34, 31, and 20, respectively) and to also compare gut mycobiota opsonization by secretory antibodies in HDs (n = 28) and patients with selective IgA deficiency (SIgAd) (n = 12). Stool mycobiota composition was determined by internal transcribed spacer gene sequencing in HDs (n = 23) and patients with SIgAd (n = 17). Circulating CD4+ T-cell cytokine secretion profiles were determined by intracellular staining. The impact of secretory IgA, purified from breast milk (n = 9), on Candidaalbicans growth and intestinal Caco-2 cell invasion was tested in vitro. RESULTS: Homeostatic IgA binds commensal fungi with a body fluid-selective pattern of recognition. In patients with SIgAd, fungal gut ecology is preserved by compensatory IgM binding to commensal fungi. Gut Calbicans overgrowth nevertheless occurs in this condition but only in clinically symptomatic patients with decreased TH17/TH22 T-cell responses. Indeed, secretory IgA can reduce in vitro budding and invasion of intestinal cells by Calbicans and therefore exert control on this pathobiont. CONCLUSION: IgA has a selective impact on Calbicans ecology to preserve fungal-host mutualism.

Antibody diversity in IVIG: Therapeutic opportunities for novel immunotherapeutic drugs.

Significant progress has been made in the elucidation of human antibody repertoires. Furthermore, non-canonical functions of antibodies have been identified that reach beyond classical functions linked to protection from pathogens. Polyclonal immunoglobulin preparations such as IVIG and SCIG represent the IgG repertoire of the donor population and will likely remain the cornerstone of antibody replacement therapy in immunodeficiencies. However, novel evidence suggests that pooled IgA might promote orthobiotic microbial colonization in gut dysbiosis linked to mucosal IgA immunodeficiency. Plasma-derived polyclonal IgG and IgA exhibit immunoregulatory effects by a diversity of different mechanisms, which have inspired the development of novel drugs. Here we highlight recent insights into IgG and IgA repertoires and discuss potential implications for polyclonal immunoglobulin therapy and inspired drugs.

Cytokines in New-Onset Refractory Status Epilepticus Predict Outcomes.

OBJECTIVE: The objective of this study was to investigate inflammation using cerebrospinal fluid (CSF) and serum cytokines/chemokines in patients with new-onset refractory status epilepticus (NORSE) to better understand the pathophysiology of NORSE and its consequences. METHODS: Patients with NORSE (n = 61, including n = 51 cryptogenic), including its subtype with prior fever known as febrile infection-related epilepsy syndrome (FIRES), were compared with patients with other refractory status epilepticus (RSE; n = 37), and control patients without SE (n = 52). We measured 12 cytokines/chemokines in serum or CSF samples using multiplexed fluorescent bead-based immunoassay detection. Cytokine levels were compared between patients with and without SE, and between the 51 patients with cryptogenic NORSE (cNORSE) and the 47 patients with a known-etiology RSE (NORSE n = 10, other RSE n = 37), and correlated with outcomes. RESULTS: A significant increase of IL-6, TNF-α, CXCL8/IL-8, CCL2, MIP-1α, and IL-12p70 pro-inflammatory cytokines/chemokines was observed in patients with SE compared with patients without SE, in serum and CSF. Serum innate immunity pro-inflammatory cytokines/chemokines (CXCL8, CCL2, and MIP-1α) were significantly higher in patients with cNORSE compared to non-cryptogenic RSE. Patients with NORSE with elevated innate immunity serum and CSF cytokine/chemokine levels had worse outcomes at discharge and at several months after the SE ended. INTERPRETATION: We identified significant differences in innate immunity serum and CSF cytokine/chemokine profiles between patients with cNORSE and non-cryptogenic RSE. The elevation of innate immunity pro-inflammatory cytokines in patients with NORSE correlated with worse short- and long-term outcomes. These findings highlight the involvement of innate immunity-related inflammation, including peripherally, and possibly of neutrophil-related immunity in cNORSE pathogenesis and suggest the importance of utilizing specific anti-inflammatory interventions. ANN NEUROL 2023;94:75-90.

T-independent responses to polysaccharides in humans mobilize marginal zone B cells prediversified against gut bacterial antigens.

Marginal zone (MZ) B cells are one of the main actors of T-independent (TI) responses in mice. To identify the B cell subset(s) involved in such responses in humans, we vaccinated healthy individuals with Pneumovax, a model TI vaccine. By high-throughput repertoire sequencing of plasma cells (PCs) isolated 7 days after vaccination and of different B cell subpopulations before and after vaccination, we show that the PC response mobilizes large clones systematically, including an immunoglobulin M component, whose diversification and amplification predated the pneumococcal vaccination. These clones could be mainly traced back to MZ B cells, together with clonally related IgA+ and, to a lesser extent, IgG+CD27+ B cells. Recombinant monoclonal antibodies isolated from large PC clones recognized a wide array of bacterial species from the gut flora, indicating that TI responses in humans largely mobilize MZ and switched B cells that most likely prediversified during mucosal immune responses against bacterial antigens and acquired pneumococcal cross-reactivity through somatic hypermutation.

Lack of association between HLA and asymptomatic SARS-CoV-2 infection.

Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B*15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B*15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the US (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B*15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified. These findings suggest that memory T-cell immunity to seasonal coronaviruses does not strongly influence the outcome of SARS-CoV-2 infection in unvaccinated individuals.

Auto-antibodies against type I IFNs in > 10% of critically ill COVID-19 patients: a prospective multicentre study.

BACKGROUND: Auto-antibodies (auto-Abs) neutralizing type I interferons (IFN) have been found in about 15% of critical cases COVID-19 pneumonia and less than 1% of mild or asymptomatic cases. Determining whether auto-Abs influence presentation and outcome of critically ill COVID-19 patients could lead to specific therapeutic interventions. Our objectives were to compare the severity at admission and the mortality of patients hospitalized for critical COVID-19 in ICU with versus without auto-Abs. RESULTS: We conducted a prospective multicentre cohort study including patients admitted in 11 intensive care units (ICUs) from Great Paris area hospitals with proven SARS-CoV-2 infection and acute respiratory failure. 925 critically ill COVID-19 patients were included. Auto-Abs neutralizing type I IFN-α2, ß and/or ω were found in 96 patients (10.3%). Demographics and comorbidities did not differ between patients with versus without auto-Abs. At ICU admission, Auto-Abs positive patients required a higher FiO2 (100% (70-100) vs. 90% (60-100), p = 0.01), but were not different in other characteristics. Mortality at day 28 was not different between patients with and without auto-Abs (18.7 vs. 23.7%, p = 0.279). In multivariable analysis, 28-day mortality was associated with age (adjusted odds ratio (aOR) = 1.06 [1.04-1.08], p < 0.001), SOFA score (aOR = 1.18 [1.12-1.23], p < 0.001) and immunosuppression (aOR = 1.82 [1.1-3.0], p = 0.02), but not with the presence of auto-Abs (aOR = 0.69 [0.38-1.26], p = 0.23). CONCLUSIONS: In ICU patients, auto-Abs against type I IFNs were found in at least 10% of patients with critical COVID-19 pneumonia. They were not associated with day 28 mortality.

Lower disease activity but higher risk of severe COVID-19 and herpes zoster in patients with systemic lupus erythematosus with pre-existing autoantibodies neutralising IFN-α.

OBJECTIVES: Type-I interferons (IFNs-I) have potent antiviral effects. IFNs-I are also overproduced in patients with systemic lupus erythematosus (SLE). Autoantibodies (AAbs) neutralising IFN-α, IFN-ß and/or IFN-ω subtypes are strong determinants of hypoxemic COVID-19 pneumonia, but their impact on inflammation remains unknown. METHODS: We retrospectively analysed a monocentric longitudinal cohort of 609 patients with SLE. Serum AAbs against IFN-α were quantified by ELISA and functionally assessed by abolishment of Madin-Darby bovine kidney cell protection by IFN-α2 against vesicular stomatitis virus challenge. Serum-neutralising activity against IFN-α2, IFN-ß and IFN-ω was also determined with a reporter luciferase activity assay. SARS-CoV-2 antibody responses were measured against wild-type spike antigen, while serum-neutralising activity was assessed against the SARS-CoV-2 historical strain and variants of concerns. RESULTS: Neutralising and non-neutralising anti-IFN-α antibodies are present at a frequency of 3.3% and 8.4%, respectively, in individuals with SLE. AAbs neutralising IFN-α, unlike non-neutralising AAbs, are associated with reduced IFN-α serum levels and a reduced likelihood to develop active disease. However, they predispose patients to an increased risk of herpes zoster and severe COVID-19 pneumonia. Severe COVID-19 pneumonia in patients with SLE is mostly associated with combined neutralisation of different IFNs-I. Finally, anti-IFN-α AAbs do not interfere with COVID-19 vaccine humoral immunogenicity. CONCLUSION: The production of non-neutralising and neutralising anti-IFN-I antibodies in SLE is likely to be a consequence of SLE-associated high IFN-I serum levels, with a beneficial effect on disease activity, yet a greater viral risk. This finding reinforces the recommendations for vaccination against SARS-CoV-2 in SLE.

Phenotypic Heterogeneity of Fulminant COVID-19--Related Myocarditis in Adults.

BACKGROUND: Adults who have been infected with SARS-CoV-2 can develop a multisystem inflammatory syndrome (MIS-A), including fulminant myocarditis. Yet, several patients fail to meet MIS-A criteria, suggesting the existence of distinct phenotypes in fulminant COVID-19-related myocarditis. OBJECTIVES: This study sought to compare the characteristics and clinical outcome between patients with fulminant COVID-19-related myocarditis fulfilling MIS-A criteria (MIS-A+) or not (MIS-A-). METHODS: A monocentric retrospective analysis of consecutive fulminant COVID-19-related myocarditis in a 26-bed intensive care unit (ICU). RESULTS: Between March 2020 and June 2021, 38 patients required ICU admission (male 66%; mean age 32 ± 15 years) for suspected fulminant COVID-19-related myocarditis. In-ICU treatment for organ failure included dobutamine 79%, norepinephrine 60%, mechanical ventilation 50%, venoarterial extracorporeal membrane oxygenation 42%, and renal replacement therapy 29%. In-hospital mortality was 13%. Twenty-five patients (66%) met the MIS-A criteria. MIS-A- patients compared with MIS-A+ patients were characterized by a shorter delay between COVID-19 symptoms onset and myocarditis, a lower left ventricular ejection fraction, and a higher rate of in-ICU organ failure, and were more likely to require mechanical circulatory support with venoarterial extracorporeal membrane oxygenation (92% vs 16%; P < 0.0001). In-hospital mortality was higher in MIS-A- patients (31% vs 4%). MIS-A+ had higher circulating levels of interleukin (IL)-22, IL-17, and tumor necrosis factor-α (TNF-α), whereas MIS-A- had higher interferon-α2 (IFN-α2) and IL-8 levels. RNA polymerase III autoantibodies were present in 7 of 13 MIS-A- patients (54%) but in none of the MIS-A+ patients. CONCLUSION: MIS-A+ and MIS-A- fulminant COVID-19-related myocarditis patients have 2 distinct phenotypes with different clinical presentations, prognosis, and immunological profiles. Differentiating these 2 phenotypes is relevant for patients' management and further understanding of their pathophysiology.

Identification of bronchoalveolar and blood immune-inflammatory biomarker signature associated with poor 28-day outcome in critically ill COVID-19 patients.

The local immune-inflammatory response elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is still poorly described, as well as the extent to which its characteristics may be associated with the outcome of critical Coronavirus disease 2019 (COVID-19). In this prospective monocenter study, all consecutive COVID-19 critically ill patients admitted from February to December 2020 and explored by fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) were included. Biological assays, including digital ELISA cytokine profiling and targeted eicosanoid metabolomic analysis, were performed on paired blood and BAL fluid (BALF). Clinical outcome was assessed through the World Health Organization 10-point Clinical Progression Scale (WHO-CPS) at the 28th day (D28) following the admission to intensive care unit. A D28-WHO-CPS value higher than 5 defined a poor outcome. Seventy-six patients were included, 45 (59%) had a poor day-28 outcome. As compared to their counterparts, patients with D28-WHO-CPS > 5 exhibited a neutrophil-predominant bronchoalveolar phenotype, with a higher BALF neutrophil/lymphocyte ratio, a blunted local type I interferon response, a decompartimentalized immune-inflammatory response illustrated by lower BALF/blood ratio of concentrations of IL-6 (1.68 [0.30-4.41] vs. 9.53 [2.56-19.1]; p = 0.001), IL-10, IL-5, IL-22 and IFN-γ, and a biological profile of vascular endothelial injury illustrated by a higher blood concentration of VEGF and higher blood and/or BALF concentrations of several vasoactive eicosanoids. In critically ill COVID-19 patients, we identified bronchoalveolar and blood immune-inflammatory biomarker signature associated with poor 28-day outcome.

Memory CD4+ T-Cell Lymphocytic Angiopathy in Fatal Forms of COVID-19 Pulmonary Infection.

The immunopathological pulmonary mechanisms leading to Coronavirus Disease (COVID-19)-related death in adults remain poorly understood. Bronchoalveolar lavage (BAL) and peripheral blood sampling were performed in 74 steroid and non-steroid-treated intensive care unit (ICU) patients (23-75 years; 44 survivors). Peripheral effector SARS-CoV-2-specific T cells were detected in 34/58 cases, mainly directed against the S1 portion of the spike protein. The BAL lymphocytosis consisted of T cells, while the mean CD4/CD8 ratio was 1.80 in non-steroid- treated patients and 1.14 in steroid-treated patients. Moreover, strong BAL SARS-CoV-2 specific T-cell responses were detected in 4/4 surviving and 3/3 non-surviving patients. Serum IFN-γ and IL-6 levels were decreased in steroid-treated patients when compared to non-steroid treated patients. In the lung samples from 3 (1 non-ICU and 2 ICU) additional deceased cases, a lymphocytic memory CD4 T-cell angiopathy colocalizing with SARS-CoV-2 was also observed. Taken together, these data show that disease severity occurs despite strong antiviral CD4 T cell-specific responses migrating to the lung, which could suggest a pathogenic role for perivascular memory CD4 T cells upon fatal COVID-19 pneumonia.

The Polarity and Specificity of Antiviral T Lymphocyte Responses Determine Susceptibility to SARS-CoV-2 Infection in Patients with Cancer and Healthy Individuals.

Vaccination against coronavirus disease 2019 (COVID-19) relies on the in-depth understanding of protective immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We characterized the polarity and specificity of memory T cells directed against SARS-CoV-2 viral lysates and peptides to determine correlates with spontaneous, virus-elicited, or vaccine-induced protection against COVID-19 in disease-free and cancer-bearing individuals. A disbalance between type 1 and 2 cytokine release was associated with high susceptibility to COVID-19. Individuals susceptible to infection exhibited a specific deficit in the T helper 1/T cytotoxic 1 (Th1/Tc1) peptide repertoire affecting the receptor binding domain of the spike protein (S1-RBD), a hotspot of viral mutations. Current vaccines triggered Th1/Tc1 responses in only a fraction of all subject categories, more effectively against the original sequence of S1-RBD than that from viral variants. We speculate that the next generation of vaccines should elicit Th1/Tc1 T-cell responses against the S1-RBD domain of emerging viral variants. SIGNIFICANCE: This study prospectively analyzed virus-specific T-cell correlates of protection against COVID-19 in healthy and cancer-bearing individuals. A disbalance between Th1/Th2 recall responses conferred susceptibility to COVID-19 in both populations, coinciding with selective defects in Th1 recognition of the receptor binding domain of spike. See related commentary by McGary and Vardhana, p. 892. This article is highlighted in the In This Issue feature, p. 873.

Pre-COVID-19 Immunity to Common Cold Human Coronaviruses Induces a Recall-Type IgG Response to SARS-CoV-2 Antigens Without Cross-Neutralisation.

The capacity of pre-existing immunity to human common coronaviruses (HCoV) to cross-protect against de novo COVID-19is yet unknown. In this work, we studied the sera of 175 COVID-19 patients, 76 healthy donors and 3 intravenous immunoglobulins (IVIG) batches. We found that most COVID-19 patients developed anti-SARS-CoV-2 IgG antibodies before IgM. Moreover, the capacity of their IgGs to react to beta-HCoV, was present in the early sera of most patients before the appearance of anti-SARS-CoV-2 IgG. This implied that a recall-type antibody response was generated. In comparison, the patients that mounted an anti-SARS-COV2 IgM response, prior to IgG responses had lower titres of anti-beta-HCoV IgG antibodies. This indicated that pre-existing immunity to beta-HCoV was conducive to the generation of memory type responses to SARS-COV-2. Finally, we also found that pre-COVID-19-era sera and IVIG cross-reacted with SARS-CoV-2 antigens without neutralising SARS-CoV-2 infectivity in vitro. Put together, these results indicate that whilst pre-existing immunity to HCoV is responsible for recall-type IgG responses to SARS-CoV-2, it does not lead to cross-protection against COVID-19.