Construction of the Enterococcal Strain Expressing Immunogenic Fragment of SARS-Cov-2 Virus

Contemporary SARS-Cov-2 pandemic, besides its dramatic global influence on the human race including health care systems, economies, and political decisions, opened a window for the global experiment with human vaccination employing novel injectable vaccines providing predominantly specific IgG response with little knowledge of their impact on the mucosal immunity. However, it is widely accepted that protection against the pathogens at the gates of the infection - on mucosal surfaces—predominantly rely on an IgA response. Some genetically modified bacteria, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Probiotic-based vaccines for mucous membranes are easy to produce in large quantities; they have low cost, provide quite a long T-cell memory, and gut IgA response to oral vaccines is highly synchronized and strongly oligoclonal. Here we present a study demonstrating construction of the novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. This DNA fragment was inserted in frame into major pili protein gene with d2 domain of enterococcal operon encoding for pili. The DNA sequencing proved the presence of the insert in enterococcal genome. RNA transcription, immunoprecipitation, and immune electron microscopy with human sera obtained from the SARS-Cov-2 patients demonstrated expression of SARS-Cov-2 antigens in bacteria. Taken together the data obtained allowed considering this genetically modified probiotic strain as an interesting candidate for vaccine against SARS-Cov-2.

Characterization of Glycosylation-Specific Systemic and Mucosal IgA Antibody Responses to Escherichia coli Mucinase YghJ (SslE)

Efforts to develop broadly protective vaccines against pathogenic Escherichia coli are ongoing. A potential antigen candidate for vaccine development is the metalloprotease YghJ, or SslE. YghJ is a conserved mucinase that is immunogenic, heavily glycosylated, and produced by most pathogenic E. coli. To develop efficacious YghJ-based vaccines, there is a need to investigate to what extent potentially protective antibody responses target glycosylated epitopes in YghJ and to describe variations in the quality of YghJ glycosylation in the E. coli population. In this study we estimated the proportion of anti-YghJ IgA antibodies that targeted glycosylated epitopes in serum and intestinal lavage samples from 21 volunteers experimentally infected with wild-type enterotoxigenic E. coli (ETEC) strain TW10722. Glycosylated and non-glycosylated YghJ was expressed, purified, and then gycosylation pattern was verified by BEMAP analysis. Then we used a multiplex bead flow cytometric assay to analyse samples from before and 10 days after TW10722 was ingested. We found that 20 (95%) of the 21 volunteers had IgA antibody responses to homologous, glycosylated YghJ, with a median fold increase in IgA levels of 7.9 (interquartile range [IQR]: 7.1, 11.1) in serum and 3.7 (IQR: 2.1, 10.7) in lavage. The median proportion of anti-YghJ IgA response that specifically targeted glycosylated epitopes was 0.45 (IQR: 0.30, 0.59) in serum and 0.07 (IQR: 0.01, 0.22) in lavage. Our findings suggest that a substantial, but variable, proportion of the IgA antibody response to YghJ in serum during ETEC infection is targeted against glycosylated epitopes, but that gut IgA responses largely target non-glycosylated epitopes. Further research into IgA targeting glycosylated YghJ epitopes is of interest to the vaccine development efforts.

Immunoglobulin (Ig)A seropositivity against SARS-CoV-2 in healthcare workers in Israel, 4 April to 13 July 2020: an observational study

Introduction The COVID-19 pandemic has put healthcare workers (HCW) at significant risk. Presence of antibodies can confirm prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Aim This study investigates the prevalence of IgA and IgG antibodies against SARS-CoV-2 in HCW. Methods Performance of IgA and IgG antibody ELISA assays were initially evaluated in positive and negative SARS-CoV-2 serum samples. IgA and IgG antibodies against SARS-CoV-2 were measured in 428 asymptomatic HCW. We assessed the risk of two groups: HCW with high exposure risk outside work (HROW) residing in areas where COVID-19 was endemic (n = 162) and HCW with high exposure risk at work (HRAW) in a COVID-19 intensive care unit (ICU) (n = 97). Results Sensitivities of 80% and 81.2% and specificities of 97.2% and 98% were observed for IgA and IgG antibodies, respectively. Of the 428 HCW, three were positive for IgG and 27 for IgA. Only 3/27 (11%) IgA-positive HCW had IgG antibodies compared with 50/62 (81%) in a group of previous SARS-CoV-2-PCR-positive individuals. Consecutive samples from IgA-positive HCW demonstrated IgA persistence 18–83 days in 12/20 samples and IgG seroconversion in 1/20 samples. IgA antibodies were present in 8.6% of HROW and 2% of HRAW. Conclusions SARS-CoV-2 exposure may lead to asymptomatic transient IgA response without IgG seroconversion. The significance of these findings needs further study. Out of work exposure is a possible risk of SARS-CoV-2 infection in HCW and infection in HCW can be controlled if adequate protective equipment is implemented.

Serology study after BTN162b2 vaccination in participants previously infected with SARS-CoV-2 in two different waves versus naïve

Background The antibody response to SARS-CoV-2 mRNA vaccines in individuals with waning immunity generated by a previous SARS-CoV-2 infection, as well as the patterns of IgA and IgM responses in previously infected and in naïve individuals are still poorly understood. Methods We performed a serology study in a cohort of BTN162b2 mRNA vaccine recipients who were immunologically naïve (N, n = 50) or had been previously infected with SARS-CoV-2 (P.I., n = 51) during the first (n = 25) or second (n = 26) pandemic waves in Italy, respectively. We measured IgG, IgM and IgA antibodies against the SARS-CoV-2 Spike (S) and IgG against the nucleocapsid (N) proteins, as well as the neutralizing activity of sera collected before vaccination, after the first and second dose of vaccine. Results Most P.I. individuals from the first pandemic wave who showed declining antibody titres responded to the first vaccine dose with IgG-S and pseudovirus neutralization titres that were significantly higher than those observed in N individuals after the second vaccine dose. In all recipients, a single dose of vaccine was sufficient to induce a potent IgA response that was not associated with serum neutralization titres. We observed an unconventional pattern of IgM responses that were elicited in only half of immunologically naïve subjects even after the second vaccine dose. Conclusions The response to a single dose of vaccine in P.I. individuals is more potent than that observed in N individuals after two doses. Vaccine-induced IgA are not associated with serum neutralization.

Anti-Interleukin Biologics for the treatment of the Atopic March Diseases

The atopic march refers to the natural history of allergic disorders as they develop from infancy to childhood. Classically, the atopic march begins with atopic dermatitis (AD), followed by food allergy, advancing to asthma, allergic rhinitis (AR), and finally to the fifth member eosinophilic esophagitis. The pathogenesis of the atopic march is complex, and involves genetic, immunological, and environmental factors. T helper type 2 (Th2) lymphocytes, and epithelial cells play a key role in the pathogenesis of the diseases in the atopic march. Th2 cells secrete cytokines, such as interleukin-5 (IL-5), IL-4, and IL-13, whereas, epithelial cell injury release alarmin cytokines, including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). Th2 cells and alarmin cytokines play an important role in the development of eczematous skin lesions, airway inflammation and remodeling, and oesophageal mucosal inflammation. Treatment of eosinophilic asthma and associated comorbid disorders is challenging, and requires a precision targeted approach with biologics. Dupilumab is a fully humanized IgG4 monoclonal antibody to the IL-4Rα, which mediates signaling to both IL-4 and IL-13, and blocks their immunopathological effects. Dupilumab is the only biologic that has been approved for the treatment of eosinophilic asthma, AD, and eosinophilic esophagitis. In patients with eosinophilic asthma treatment with dupilumab has been shown to improve asthma control, reduce exacerbations, and improve lung function. In patients with atopic dermatitis dupilumab has been demonstrated to improve the Eczema Area Severity Index (EASI) score, Investigator’s Global Assessment (IGA) response, SCORing Atopic Dermatitis (SCORAD) score, and the Peak Pruritus Numerical Rating (PNR) scale. Lebrikizumab and Tralokinumab (anti-IL-13) failed to show the expected results for the treatment of asthma, astoundingly, in several clinical trials they have been shown to significantly improvement EASI score, IGA response, SCORAD score, PNR scale, sleep architecture, and Dermatology Life Quality Index (DLQI). They have been granted First Tract Designation, and European Commission regulatory approval, respectively. Tezepelumab (anti-TSLP) is approved for the treatment of eosinophilic asthma, and has been shown to significantly reduce exacerbations, and improve asthma control, lung function, and HLQoL. However, tezepelumab did not meet the endpoints in phase II for the treatment of AD. Keywords: Atopic March; Atopic Dermatitis; Eosinophilic Asthma; Interleukin; Dupilumab; Tralokinumab

Efficacy and Safety of Janus Kinase Inhibitors for the Treatment of Atopic Dermatitis: A Systematic Review and Meta-Analysis

<b><i>Background:</i></b> Current therapeutic options for atopic dermatitis (AD) are limited. Janus kinase (JAK) inhibitors may be viable alternatives. <b><i>Objectives:</i></b> To assess the efficacy and safety of JAK inhibitors for AD treatment. <b><i>Methods:</i></b> We searched PubMed, Embase, the Cochrane Controlled Register of Trials, Web of Science, Global Resource of Eczema Trials database, and ClinicalTrials.gov from inception to September 1, 2020. Randomized clinical trials (RCTs) comparing JAK inhibitors with placebo/vehicle treatment for AD patients were included. The primary study outcomes included (1) the change (%) from the Eczema Area and Severity Index (EASI) baseline expressed as weighted mean difference (WMD) and 95% confidence interval (95% CI), and (2) the Investigator’s Global Assessment (IGA) response and safety outcomes expressed as relative risk (RR) and 95% CI. <b><i>Results:</i></b> We included 14 RCTs published in 13 studies (3,822 patients). Treatment with JAK inhibitors significantly improved IGA response (RR 2.83, 95% CI 2.25–3.56, <i>p</i> &#x3c; 0.001) and EASI score (WMD –28.82, 95% CI –34.48 to −23.16, <i>p</i> &#x3c; 0.001). JAK inhibitor treatment achieved the largest improvement in both IGA response (RR 3.59, 95% CI 2.66–4.84, <i>p</i> &#x3c; 0.001) and EASI score (WMD –42.00, 95% CI –48.64 to −35.36, <i>p</i> &#x3c; 0.001) by week 4 of treatment. Topical JAK inhibitors were significantly more efficacious than oral inhibitors. Upadacitinib treatment for 4 weeks was most effective in reducing EASI score (WMD –53.92, 95% CI –69.26 to −38.58, <i>p</i> &#x3c; 0.001), while abrocitinib for 4 weeks led to the most effective IGA response (RR 5.47, 95% CI 2.74–10.93, <i>p</i> &#x3c; 0.001). There was no difference in the frequency of adverse events (AEs) leading to discontinuation; however, JAK inhibitors use, especially abrocitinib, led to a higher incidence of treatment-emergent AEs (RR 1.25, 95% CI 1.10–1.42, <i>p</i> = 0.001). <b><i>Conclusion:</i></b> Our results imply that JAK inhibitors are an effective and safe AD treatment. Nevertheless, further trials with longer duration and head-to-head comparisons of different JAK inhibitors are needed.

Axial spondyloarthritis patients have altered mucosal IgA response to oral and fecal microbiota

Objective: To investigate whether axial spondyloarthritis (AxSpA) patients have an altered immunoglobulin A (IgA) response in the gut and oral microbial communities. Methods: We performed 16S rRNA gene (16S) sequencing on IgA positive (IgA+) and IgA negative (IgA-) fractions (IgA-SEQ) from feces (n=17 AxSpA; n=14 healthy) and saliva (n=17 AxSpA; n=12 healthy), as well as on IgA-unsorted fecal and salivary samples. PICRUSt2 was used to predict microbial metabolic potential in AxSpA patients and healthy controls (HCs). Results: IgA-SEQ revealed enrichment of several microbes in the fecal (Akkermansia, Ruminococcaceae, Lachnospira) and salivary (Prevotellaceae, Actinobacillus) microbiome in AxSpA patients as compared with HCs. Fecal microbiome from AxSpA patients showed a trend towards increased alpha diversity of the IgA+ fraction and decreased diversity in the IgA- fraction in comparison with HCs, while the salivary microbiome exhibits a significant decrease in alpha diversity in both IgA+ and IgA- fractions. Increased IgA coating of Clostridiales Family XIII correlated with disease severity. Inferred metagenomic analysis suggests perturbation of metabolites and metabolic pathways for inflammation (oxidative phosphorylation, glutathione metabolism) and metabolism (propanoate and butanoate metabolism) in AxSpA patients. Conclusions: Analyses of fecal and salivary microbes from AxSpA patients reveal distinct populations of immunoreactive microbes using novel IgA-SEQ approach, which were not captured by comparing their relative abundance with HCs. Predictive metagenomic analysis revealed perturbation of metabolites/metabolic pathways in AxSpA patients. Future studies on these immunoreactive microbes may lead to better understanding of the functional role of IgA in maintaining microbial structure and human health.