A linear B-cell epitope close to the furin cleavage site within the S1 domain of SARS-CoV-2 Spike protein discriminates the humoral immune response of nucleic acid- and protein-based vaccine cohorts.

Background: Understanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants. Methods: We used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA. Results: Overall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees. Conclusions: Understanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design.

Immune profiling of SARS-CoV-2 epitopes in asymptomatic and symptomatic pediatric and adult patients.

BACKGROUND: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients. METHODS: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5). RESULTS: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels. CONCLUSIONS: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection.

Characterisation and analysis of linear epitopes corresponding to SARS-CoV-2 outbreak in Jilin Province, China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants have caused hundreds of thousands of deaths and shown serious social influence worldwide. Jilin Province, China, experienced the first wave of the outbreak from December 2020 to February 2021. Here, we analysed the genomic characteristics of the SARS-CoV-2 outbreak in Jilin province using a phylogeographic tree and found that clinical isolates belonged to the B.1 lineage, which was considered to be the ancestral lineage. Several dominant SARS-CoV-2 specific linear B cell epitopes that reacted with the convalescent sera were also analysed and identified using a peptide microarray composed of S, M, and E proteins. Moreover, the serum of convalescent patients infected with SARS-CoV-2 showed neutralising activity against four widely spreading SARS-CoV-2 variants; however, significant differences were observed in neutralising activities against different SARS-CoV-2 variants. These data provide important information on genomic characteristics, linear epitopes, and neutralising activity of SARS-CoV-2 outbreak in Jilin Province, China, which may aid in understanding disease patterns and regional aspects of the pandemic. This article is protected by copyright. All rights reserved.

Humoral Immune Response Profile of COVID-19 Reveals Severity and Variant-Specific Epitopes: Lessons from SARS-CoV-2 Peptide Microarray.

The amaranthine scale of the COVID-19 pandemic and unpredictable disease severity is of grave concern. Serological diagnostic aids are an excellent choice for clinicians for rapid and easy prognosis of the disease. To this end, we studied the humoral immune response to SARS-CoV-2 infection to map immunogenic regions in the SARS-CoV-2 proteome at amino acid resolution using a high-density SARS-CoV-2 proteome peptide microarray. The microarray has 4932 overlapping peptides printed in duplicates spanning the entire SARS-CoV-2 proteome. We found 204 and 676 immunogenic peptides against IgA and IgG, corresponding to 137 and 412 IgA and IgG epitopes, respectively. Of these, 6 and 307 epitopes could discriminate between disease severity. The emergence of variants has added to the complexity of the disease. Using the mutation panel available, we could detect 5 and 10 immunogenic peptides against IgA and IgG with mutations belonging to SAR-CoV-2 variants. The study revealed severity-based epitopes that could be presented as potential prognostic serological markers. Further, the mutant epitope immunogenicity could indicate the putative use of these markers for diagnosing variants responsible for the infection.

Applications of Peptide Microarrays in Autoantibody, Infection, and Cancer Detection

The diversity of the antigen-specific humoral immune response reflects the interaction of the immune system with pathogens and autoantigens. Peptide microarray analysis opens up new perspectives for the use of antibodies as diagnostic biomarkers and provides unique access to a more differentiated view on humoral responses to disease. This review focuses on the latest applications of peptide microarrays for the serologic medical diagnosis of autoimmunity, infectious diseases (including COVID-19), and cancer.

ANTI-TNF THERAPY for IMMUNE MEDIATED INFLAMMATORY DISEASES MAY BE ASSOCIATED with LOWER ANTIBODY LEVELS and VIRUS NEUTRALIZATION EFFICACY FOLLOWING SARS-COV-2 MRNA VACCINATION

Background: The impact of immunosuppressants on COVID-19 vaccination response and durability in patients with immune-mediated infammatory diseases (IMID) is yet to be fully characterized. Humoral response may be attenuated in these patients especially those on B cell depleting therapy and higher doses of corticosteroids, but data regarding other immunosuppressants are scarce. Objectives: We aimed to investigate antibody and T cell responses and durability to SARS-CoV-2 mRNA vaccines (BNT162b and/or mRNA 1273) in IMID patients on immunomodulatory maintenance therapy other than B-cell depleting therapy and corticosteroids. Methods: This prospective observational cohort study examined the immuno-genicity of SARS-CoV-2 mRNA vaccines in adult patients with IMIDs (psoriatic arthritis, psoriasis, infammatory bowel disease and rheumatoid arthritis) with or without maintenance immunosuppressive therapies (anti-TNF, methotrexate/azathioprine [MTX/AZA], anti-TNF + MTX/AZA, anti IL12/23, anti-IL-17, anti-IL23) compared to healthy controls. Automated ELISA for IgGs to spike trimer, spike receptor binding domain (RBD) and the nucleocapsid (NP) and T-cell release of 9 cytokines (IFNg, IL2, IL4, IL17A, TNF) and cytotoxic molecules (sFasL, GzmA, GzmB, Perforinin) in cell culture supernatants following stimulation with spike or NP peptide arrays were conducted at 4 time points: T1=pre vaccination, T2=me-dian 26 days after dose 1, T3=median 16 days after dose 2 and T4=median 106 days after dose 2. Neutralization assays against four SARS-CoV-2 variants (wild type, delta, beta and gamma) were conducted at T3. Results: We followed 150 subjects: 26 healthy controls and 124 IMID patients: 9 untreated, 44 on anti-TNF, 16 on anti-TNF with MTX/AZA, 10 on anti-IL23, 28 on anti-IL12/23, 9 on anti-IL17, 8 on MTX/AZA (Table 1). Most patients mounted antibody and T cell responses with increases from dose 1 to dose 2 (100% sero-conversion at T3) and some decline by T4, with variability within groups. Antibody levels and neutralization efficacy was lower in anti-TNFgroups (anti-TNF, anti-TNF + MTX/AZA) compared to controls and waned by T4 (Figure 1). T cell responses were not consistently different between groups. Pooled data showed a higher antibody response to mRNA-1273 compared to BNT162b. Conclusion: Following 2 doses of mRNA vaccination there is 100% seroconver-sion in IMID patients on maintenance therapy. Antibody levels and neutralization efficacy in anti-TNF group are lower than controls, and wane substantially by 3 months after dose 2. These fndings highlight the need for third dose in patients undergoing treatment with anti-TNF therapy and continued monitoring of immunity in these patient groups, taking into consideration newer variants and additional vaccine doses.

Longitudinal immune profiling reveals dominant epitopes mediating long-term humoral immunity in COVID-19-convalescent individuals.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic and contagious coronavirus that caused a global pandemic with 5.2 million fatalities to date. Questions concerning serologic features of long-term immunity, especially dominant epitopes mediating durable antibody responses after SARS-CoV-2 infection, remain to be elucidated. OBJECTIVE: We aimed to dissect the kinetics and longevity of immune responses in coronavirus disease 2019 (COVID-19) patients, as well as the epitopes responsible for sustained long-term humoral immunity against SARS-CoV-2. METHODS: We assessed SARS-CoV-2 immune dynamics up to 180 to 220 days after disease onset in 31 individuals who predominantly experienced moderate symptoms of COVID-19, then performed a proteome-wide profiling of dominant epitopes responsible for persistent humoral immune responses. RESULTS: Longitudinal analysis revealed sustained SARS-CoV-2 spike protein-specific antibodies and neutralizing antibodies in COVID-19 patients, along with activation of cytokine production at early stages after SARS-CoV-2 infection. Highly reactive epitopes that were capable of mediating long-term antibody responses were shown to be located at the spike and ORF1ab proteins. Key epitopes of the SARS-CoV-2 spike protein were mapped to the N-terminal domain of the S1 subunit and the S2 subunit, with varying degrees of sequence homology among endemic human coronaviruses and high sequence identity between the early SARS-CoV-2 (Wuhan-Hu-1) and current circulating variants. CONCLUSION: SARS-CoV-2 infection induces persistent humoral immunity in COVID-19-convalescent individuals by targeting dominant epitopes located at the spike and ORF1ab proteins that mediate long-term immune responses. Our findings provide a path to aid rational vaccine design and diagnostic development.

Peptide microarray-based analysis of antibody responses to SARS-CoV-2 identifies unique epitopes with potential for diagnostic test development.

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n = 24 patient samples and n = 12 control samples were screened for antibodies against the entire SARS-CoV-2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, OC43, and 229E. While widespread cross-reactivity was revealed across several immunodominant regions of S and N, IgG binding to several SARS-CoV-2-derived peptides provided statistically significant discrimination between COVID-19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID-19-specific diagnostic antibody tests.