The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence1-3. Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

Clinical outcome and component-specific antibody levels in egg allergic children after lightened oral immunotherapy

Objective: To evaluate the clinical outcome of lightened version of egg oral immunotherapy (OIT) and to analyze egg allergen component-specific antibody levels during short up-dosing with egg white powder and maintenance by egg in daily diet. Patients and methods: Eighteen egg-allergic children received egg powder with short up--dosing and they maintained tolerance using egg in daily diet. Seventeen egg-allergic children served as a control group. Component-resolved analysis of serum immunoglobulin E (IgE), IgA1, IgA2, and IgG4 levels were determined at inclusion, after up-dosing and after 1 year of immunotherapy. Skin-prick tests were performed at inclusion and after 1 year of therapy. Results: All 18 patients in the egg OIT group were successfully desensitized. Desensitization was achieved on average in 4.5 months. In the control group, only two children tolerated egg in oral food challenge after 1 year. Of the measured immune markers, smaller wheal diameters in skin-prick testing, reduction in component-specific IgE levels, and increase in component-specific IgA1, IgA2, and IgG4 levels were associated with desensitization. Conclusion: A lightened egg OIT is effective and safe in children with egg allergy. Increase in all egg component-specific IgA1, IgA2 and IgG4 levels and decrease in all egg component--specific IgE levels were observed after 12 months of OIT (AU)

Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization.

The COVID-19 pandemic has fostered major advances in vaccination technologies1-4; however, there are urgent needs for vaccines that induce mucosal immune responses and for single-dose, non-invasive administration4-6. Here we develop an inhalable, single-dose, dry powder aerosol SARS-CoV-2 vaccine that induces potent systemic and mucosal immune responses. The vaccine encapsulates assembled nanoparticles comprising proteinaceous cholera toxin B subunits displaying the SARS-CoV-2 RBD antigen within microcapsules of optimal aerodynamic size, and this unique nano-micro coupled structure supports efficient alveoli delivery, sustained antigen release and antigen-presenting cell uptake, which are favourable features for the induction of immune responses. Moreover, this vaccine induces strong production of IgG and IgA, as well as a local T cell response, collectively conferring effective protection against SARS-CoV-2 in mice, hamsters and nonhuman primates. Finally, we also demonstrate a mosaic iteration of the vaccine that co-displays ancestral and Omicron antigens, extending the breadth of antibody response against co-circulating strains and transmission of the Omicron variant. These findings support the use of this inhaled vaccine as a promising multivalent platform for fighting COVID-19 and other respiratory infectious diseases.

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Patterns of IgA Autoantibody Generation, Inflammatory Responses and Extracellular Matrix Metabolism in Patients with Alcohol Use Disorder.

Recent data have emphasized the role of inflammation and intestinal immunoglobulin A (IgA) responses in the pathogenesis of alcoholic liver disease (ALD). In order to further explore such associations, we compared IgA titers against antigens targeted to ethanol metabolites and tissue transglutaminase with pro- and anti-inflammatory mediators of inflammation, markers of liver status, transferrin protein desialylation and extracellular matrix metabolism in alcohol-dependent patients with or without liver disease and in healthy controls. Serum IgAs against protein adducts with acetaldehyde (HbAch-IgA), the first metabolite of ethanol, and tissue transglutaminase (tTG-IgA), desialylated transferrin (CDT), pro- and anti-inflammatory cytokines, markers of liver status (GT, ALP) and extracellular matrix metabolism (PIIINP, PINP, hyaluronic acid, ICTP and CTx) were measured in alcohol-dependent patients with (n = 83) or without (n = 105) liver disease and 88 healthy controls representing either moderate drinkers or abstainers. In ALD patients, both tTG-IgA and HbAch-IgA titers were significantly higher than those in the alcoholics without liver disease (p < 0.0005 for tTG-IgA, p = 0.006 for Hb-Ach-IgA) or in healthy controls (p < 0.0005 for both comparisons). The HbAch-IgA levels in the alcoholics without liver disease also exceeded those found in healthy controls (p = 0.0008). In ROC analyses, anti-tTG-antibodies showed an excellent discriminative value in differentiating between ALD patients and healthy controls (AUC = 0.95, p < 0.0005). Significant correlations emerged between tTG-IgAs and HbAch-IgAs (rs = 0.462, p < 0.0005), CDT (rs = 0.413, p < 0.0001), GT (rs = 0.487, p < 0.0001), alkaline phosphatase (rs = 0.466, p < 0.0001), serum markers of fibrogenesis: PIIINP (rs = 0.634, p < 0.0001), hyaluronic acid (rs = 0.575, p < 0.0001), ICTP (rs = 0.482, p < 0.0001), pro-inflammatory cytokines IL-6 (rs = 0.581, p < 0.0001), IL-8 (rs = 0.535, p < 0.0001) and TNF-α (rs = 0.591, p < 0.0001), whereas significant inverse correlations were observed with serum TGF-ß (rs = -0.366, p < 0.0001) and CTx, a marker of collagen degradation (rs = -0.495, p < 0.0001). The data indicate that the induction of IgA immune responses toward ethanol metabolites and tissue transglutaminaseis a characteristic feature of patients with AUD and coincides with the activation of inflammation, extracellular matrix remodeling and the generation of aberrantly glycosylated proteins. These processes appear to work in concert in the sequence of events leading from heavy drinking to ALD.

Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Budesonide delayed-release capsules to reduce proteinuria in adults with primary immunoglobulin A nephropathy.

INTRODUCTION: Immunoglobulin A nephropathy (IgAN) is characterized by mesangial deposition of immune complexes containing galactose-deficient IgA1 (Gd-IgA1). This Gd-IgA1 is believed to originate from mucosally sited B cells, which are abundant in the Peyer's patches-rich distal ileum. Nefecon is a targeted-release form of budesonide developed to act in the distal ileum, thereby exerting a direct action on the mucosal tissue implicated in the pathogenesis of the disease. AREAS COVERED: This review discusses IgAN pathophysiology and provides an overview of the current therapeutic landscape, focusing on Nefecon, the first drug to receive accelerated US approval and conditional EU approval for the treatment of patients with IgAN at risk of rapid disease progression. EXPERT OPINION: Nefecon trial data thus far have demonstrated a promising efficacy profile, with a predictable pattern of adverse events. Treatment with Nefecon for 9 months reduces proteinuria substantially (Part A of the Phase 3 trial and the Phase 2b trial). A nearly complete prevention of deterioration of renal function has been observed at 12 months in patients at greatest risk of rapid disease progression. Long-term data from Part B of the Phase 3 study will provide 24-month data, furthering understanding of the durability of the 9-month treatment course.

[The reasons for the formation of tolerance to food antigens].

One of the main issues of the peculiarities of the immune reactions of the gastrointestinal tract is the mechanisms of ensuring tolerance to food antigens. Concentrations of antibodies to food antigens actually reflect the state of the intestinal mucosa barrier function, and the degree of penetration of antigens into the blood determines the level of immune response to them. The aim of the study was to determine the risk criteria for violation of tolerance to food antigens. Material and methods. The study included the results of a survey and examination of 1334 adults living in the north of the European part of the Russian Federation, including 1100 born in the North, of which 970 were women and 364 were men. The average age of the respondents was 45.5±1.0 years. The comparison group consisted of 344 patients with pathology of the gastrointestinal tract who applied to the medical company "Biocor". The content of immunoglobulins (Ig) G to food antigens, total IgA, cytokines (tumor necrosis factor α, interleukin-6, interleukin-4) in blood serum were determined by enzyme immunoassay. Results. Rural residents often (more than 28%) have elevated concentrations of IgG to potato, river fish, wheat and rye antigens. Urban residents have the most pronounced decrease in tolerance to food antigens of chicken, cod, beef and pork. In healthy individuals, elevated (>100 ME/ml) concentrations of antibodies to meat products are recorded in the range of 11.3-13.9%, to dairy antigens - 11.5-14.1%, cereals - 11.9-13.4%. Slightly less frequently, elevated concentrations of antibodies to fish antigens (7.5-10.1%), vegetables (3.8-7.0%) and fruits (4.9-6.5%) are detected. In inflammatory and oncological diseases of the gastrointestinal tract, the content of antibodies to food antigens increases sharply. On average, the frequency of impaired tolerance to food antigens in patients is 2.7-6.1 times higher than in healthy individuals. Conclusion. Violation of tolerance to food antigens is associated with an increase in blood pro-inflammatory cytokines, mainly interleukin-6. In practically healthy individuals, a decrease in tolerance to food antigens is associated with a deficiency of blood IgA. The risk criteria for violation of the diet or consumption of low-quality foods may be an increase in the frequency of detection of elevated concentrations of antibodies to meat products in 14.6±3.0%, fish - 10.7±2.3%, cereals - 13.7±1.6%, dairy products - 14.8±1.5%, vegetables - 7.8±2.4% and fruits - 6.9±5.8%.

Salivary and serum IgA and IgG responses to SARS-CoV-2-spike protein following SARS-CoV-2 infection and after immunization with COVID-19 vaccines.

Background: Secretory immunoglobulin A (sIgA) plays an important role in antiviral protective immunity. Although salivary testing has been used for many viral infections, including severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), its use has not yet been well established with the SARS coronavirus 2 (SARS-CoV-2). Quantification of salivary IgA and IgG antibodies can elucidate mucosal and systemic immune responses after natural infection or vaccination. Here, we report the development and validation of a rapid enzyme-linked immunosorbent assay (ELISA) for anti-SARS-CoV-2 salivary IgA and serum IgG antibodies, and present quantitative results for immunized subjects both prior to or following COVID-19 infections. Objective: Total and serum SARS-CoV-2 spike-specific IgG responses were compared with salivary spike-specific IgA and IgG responses in samples obtained from patients recently infected with SARS-CoV-2 and from subjects recently immunized with COVID-19 vaccines. Methods: A total of 52 paired saliva and serum samples were collected from 26 study participants: 7 subjects after COVID-19 infection and 19 subjects who were uninfected. The ELISA results from these samples were compared with five prepandemic control serum samples. Total IgG and SARS-CoV-2 spike-specific IgG in the serum samples from the subjects who were infected and vaccinated were also measured in a commercial laboratory with an enzyme immunoassay. Results: A wide variation in antibody responses was seen in salivary and serum samples measured by both methods. Three groups of serum total and IgG spike-specific SARS-CoV-2 antibody responses were observed: (1) low, (2) intermediate, and (3) high antibody responders. A correlational analysis of salivary IgA (sIgA) responses with serum IgG concentrations showed a statistical correlation in the low and intermediate antibody responder groups but not in the high group (which we believe was a result of saturation). Conclusion: These preliminary findings suggest measuring salivary and serum IgG and IgA merit further investigation as markers of current or recent SARS-CoV-2 infections.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

[C1q or IgA deposition in glomeruli of children with primary membranous nephropathy].

Objective: To assess the correlation of glomerular C1q or IgA deposition with clinical and pathological features of primary membranous nephropathy (PMN) in children. Methods: The clinical and pathological manifestations including (phospholipase A2 receptor, PLA2R) and IgG subclasses staining in renal biopsies, serum anti-PLA2R antibody and therapeutic response of 33 children diagnosed with PMN in Peking University First Hospital from December 2012 to December 2020 were retrospectively summarized and analyzed. According to results of PLA2R test and findings renal pathological, the patients were divided into PLA2R-related group and non-PLA2R-related group, typical MN group and atypical MN group, C1q deposit group and non-C1q deposit group, as well as IgA deposit group and non-IgA deposit group respectively. T-test, Mann-Whitney U test and Fisher's exact probability test were used for comparison between the groups. Results: Among the 33 children with PMN, there were 20 males and 13 females, of that the age of onset was 11 (8, 13) years, and 32 patients had nephrotic level proteinuria. Renal biopsies were performed at 4.6 (2.1, 11.6) months after onset, and 28 patients (85%) received glucocorticoid or immunosuppressive therapy prior to renal biopsy. There were 20 cases (61%) with PLA2R-related MN and 13 cases (39%) with non-PLA2R-related MN. Compared with the non-PLA2R-related group, the PLA2R-related group had an older age of onset (12 (10, 13) vs. 7 (3, 12) years, Z=-2.52, P=0.011), a lower preceding infection rate (45% (9/20) vs. 11/13, P=0.032) and lower spontaneous remission rate (0 vs. 4/13, P=0.017). Renal PLA2R positivity was significantly associated with predominant or co-deposition of IgG4 (13/17 vs. 5/15, P=0.031) and low albumin levels at renal biopsy ((25±6) vs. (29±7) g/L, t=2.14, P=0.041). There were 12 patients with typical PMN and 21 patients with atypical PMN, and no significant difference in clinical and pathological manifestations was found between these 2 groups (all P>0.05). There were 10 cases (32.3%) with glomerular C1q deposition, and their disease course before renal biopsy was significantly shorter than those without C1q deposition (1.8 (0.8, 5.9) vs. 6.0 (2.5, 22.3) months, Z=-2.27, P=0.023). Twelve cases (36.4%) had glomerular IgA deposition, and their course of disease,clinical and pathological manifestations were not significantly different from those without IgA deposition (all P>0.05). Conclusion: Glomerular C1q or IgA deposition may not affect the clinical manifestations, glomerular PLA2R and IgG subclasses staining pattern, or the response to treatment of PMN in children.

Peyer's patch TH17 cells are dispensable for gut IgA responses to oral immunization.

T helper 17 (TH17) cells located at the Peyer's patch (PP) inductive site and at the lamina propria effector site of the intestinal immune system are responsive to both pathogenic and commensal bacteria. Their plasticity to convert into follicular helper T (TFH) cells has been proposed to be central to gut immunoglobulin A (IgA) responses. Here, we used an IL-17A fate reporter mouse and an MHC-II tetramer to analyze antigen-specific CD4+ T cell subsets and isolate them for single-cell RNA sequencing after oral immunization with cholera toxin and ovalbumin. We found a TFH-dominated response with only rare antigen-specific TH17 cells (<8%) in the PP. A clonotypic analysis provided little support that clonotypes were shared between TFH and TH17 cells, arguing against TH17 plasticity as a major contributor to TFH differentiation. Two mouse models of TH17 deficiency confirmed that gut IgA responses to oral immunization do not require TH17 cells, with CD4CreRorcfl/fl mice exhibiting normal germinal centers in PP and unperturbed total IgA production in the intestine.

Enteric viruses replicate in salivary glands and infect through saliva.

Enteric viruses like norovirus, rotavirus and astrovirus have long been accepted as spreading in the population through fecal-oral transmission: viruses are shed into feces from one host and enter the oral cavity of another, bypassing salivary glands (SGs) and reaching the intestines to replicate, be shed in feces and repeat the transmission cycle1. Yet there are viruses (for example, rabies) that infect the SGs2,3, making the oral cavity one site of replication and saliva one conduit of transmission. Here we report that enteric viruses productively and persistently infect SGs, reaching titres comparable to those in the intestines. We demonstrate that enteric viruses get released into the saliva, identifying a second route of viral transmission. This is particularly significant for infected infants, whose saliva directly transmits enteric viruses to their mothers' mammary glands through backflow during suckling. This sidesteps the conventional gut-mammary axis route4 and leads to a rapid surge in maternal milk secretory IgA antibodies5,6. Lastly, we show that SG-derived spheroids7 and cell lines8 can replicate and propagate enteric viruses, generating a scalable and manageable system of production. Collectively, our research uncovers a new transmission route for enteric viruses with implications for therapeutics, diagnostics and importantly sanitation measures to prevent spread through saliva.

Early human B cell signatures of the primary antibody response to mRNA vaccination.

Messenger RNA (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective at inducing protective immunity. However, weak antibody responses are seen in some individuals, and cellular correlates of immunity remain poorly defined, especially for B cells. Here we used unbiased approaches to longitudinally dissect primary antibody, plasmablast, and memory B cell (MBC) responses to the two-dose mRNA-1273 vaccine in SARS-CoV-2-naive adults. Coordinated immunoglobulin A (IgA) and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses but earlier and more intensely after dose 2. While antibody and B cell cellular responses were generally robust, they also varied within the cohort and decreased over time after a dose-2 peak. Both antigen-nonspecific postvaccination plasmablast frequency after dose 1 and their spike-specific counterparts early after dose 2 correlated with subsequent antibody levels. This correlation between early plasmablasts and antibodies remained for titers measured at 6 months after vaccination. Several distinct antigen-specific MBC populations emerged postvaccination with varying kinetics, including two MBC populations that correlated with 2- and 6-month antibody titers. Both were IgG-expressing MBCs: one less mature, appearing as a correlate after the first dose, while the other MBC correlate showed a more mature and resting phenotype, emerging as a correlate later after dose 2. This latter MBC was also a major contributor to the sustained spike-specific MBC response observed at month 6. Thus, these plasmablasts and MBCs that emerged after both the first and second doses with distinct kinetics are potential determinants of the magnitude and durability of antibodies in response to mRNA-based vaccination.

Ultrasensitive detection of salivary SARS-CoV-2 IgG antibodies in individuals with natural and COVID-19 vaccine-induced immunity.

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3-97.2%) compared to 91.7% (95% CI 77.5-98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3-100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AIpost/pre). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgGpost/pre). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay.

SerumCovid database: Description and preliminary analysis of serological COVID-19 diagnosis in healthcare workers.

Serological databases represent an important source of information to perceive COVID-19 impact on health professionals involved in combating the disease. This paper describes SerumCovid, a COVID-19 serological database focused on the diagnosis of health professionals, providing a preliminary analysis to contribute to the understanding of the antibody response to the SARS-CoV-2. The study population comprises 321 samples from 236 healthcare and frontline workers fighting COVID-19 in Vitória de Santo Antão, Brazil. Samples were collected from at least six days of symptoms to more than 100 days. The used immunoenzymatic assays were Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA. The most common gender in SerumCovid is female, while the most common age group is between 30 and 39 years old. However, no statistical differences were observed in either genders or age categories. The most reported symptoms were fatigue, headaches, and myalgia. Still, some subjects presented positive results for IgA after 130 days. Based on a temporal analysis, we have not identified general patterns as subjects presented high and low values of IgA and IgG with different evolution trends. Unexpectedly, for subjects with both serological tests, the outcome of IgA and IgG tests were the same (either positive or negative) for more than 80% of the samples. Therefore, SerumCovid helps better understand how COVID-19 affected healthcare and frontline workers, which increases knowledge about the infection and enables direct prevention actions.

Anti-SARS-CoV-2 IgG and IgA antibodies in COVID-19 convalescent plasma do not enhance viral infection.

The novel coronavirus, SARS-CoV-2 that causes COVID-19 has resulted in the death of nearly 4 million people within the last 18 months. While preventive vaccination, and monoclonal antibody therapies have been rapidly developed and deployed, early in the pandemic the use of COVID-19 convalescent plasma (CCP) was a common means of passive immunization with a theoretical risk of antibody-dependent enhancement (ADE) of viral infection. Though vaccines elicit a strong and protective immune response and transfusion of CCP with high titers of neutralization activity are correlated with better clinical outcomes, the question of whether antibodies in CCP can enhance infection of SARS-CoV-2 has not been directly addressed. In this study, we analyzed for and observed passive transfer of neutralization activity with CCP transfusion. Furthermore, to specifically understand if antibodies against the spike protein (S) enhance infection, we measured the anti-S IgG, IgA, and IgM responses and adapted retroviral-pseudotypes to measure virus neutralization with target cells expressing the ACE2 virus receptor and the Fc alpha receptor (FcαR) or Fc gamma receptor IIA (FcγRIIA). Whereas neutralizing activity of CCP correlated best with higher titers of anti-S IgG antibodies, the neutralizing titer was not affected when Fc receptors were present on target cells. These observations support the absence of antibody-dependent enhancement of infection (ADE) by IgG and IgA isotypes found in CCP. The results presented, therefore, not only supports the therapeutic use of currently available antibody-based treatment, including the continuation of CCP transfusion strategies, but also the use of various vaccine platforms in a prophylactic approach.