Flow Cytometry-Based Measurement of Antibodies Specific for Cell Surface-Expressed Folded SARS-CoV-2 Receptor-Binding Domains.

BACKGROUND: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now become endemic and is currently one of the important respiratory virus infections regularly affecting mankind. The assessment of immunity against SARS-CoV-2 and its variants is important for guiding active and passive immunization and SARS-CoV-2-specific treatment strategies. METHODS: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron. RESULTS: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies. CONCLUSION: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern.

Vaccine Based on Recombinant Fusion Protein Combining Hepatitis B Virus PreS with SARS-CoV-2 Wild-Type- and Omicron-Derived Receptor Binding Domain Strongly Induces Omicron-Neutralizing Antibodies in a Murine Model.

BACKGROUND: COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. OBJECTIVES: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. METHODS: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum-hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. RESULTS: Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. CONCLUSION: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Recombinant PreS-fusion protein vaccine for birch pollen and apple allergy.

BACKGROUND: IgE cross-sensitization to major birch pollen allergen Bet v 1 and pathogenesis-related (PR10) plant food allergens is responsible for the pollen-food allergy syndrome. METHODS: We designed a recombinant protein, AB-PreS, consisting of non-allergenic peptides derived from the IgE-binding sites of Bet v 1 and the cross-reactive apple allergen, Mal d 1, fused to the PreS domain of HBV surface protein as immunological carrier. AB-PreS was expressed in E. coli and purified by chromatography. The allergenic and inflammatory activity of AB-PreS was tested using basophils and PBMCs from birch pollen allergic patients. The ability of antibodies induced by immunization of rabbits with AB-PreS and birch pollen extract-based vaccines to inhibit allergic patients IgE binding to Bet v 1 and Mal d 1 was assessed by ELISA. RESULTS: IgE-binding experiments and basophil activation test revealed the hypoallergenic nature of AB-PreS. AB-PreS induced lower T-cell activation and inflammatory cytokine production in cultured PBMCs from allergic patients. IgG antibodies induced by five injections with AB-PreS inhibited allergic patients' IgE binding to Bet v 1 and Mal d 1 better than did IgG induced by up to 30 injections of six licensed birch pollen allergen extract-based vaccines. Additionally, immunization with AB-PreS induced HBV-specific antibodies potentially protecting from infection with HBV. CONCLUSION: The recombinant AB-PreS-based vaccine is hypoallergenic and superior over currently registered allergen extract-based vaccines regarding the induction of blocking antibodies to Bet v 1 and Mal d 1 in animals.

Prospective assessment of pre-existing and de novo anti-HLA IgE in kidney, liver, lung and heart transplantation.

Introduction: Antibody mediated rejection (ABMR) is a major factor limiting outcome after organ transplantation. Anti-HLA donor-specific antibodies (DSA) of the IgG isotype are mainly responsible for ABMR. Recently DSA of the IgE isotype were demonstrated in murine models as well as in a small cohort of sensitized transplant recipients. In the present study, we aimed to determine the frequency of pre-existing and de novo anti-HLA IgE antibodies in a cohort of 105 solid organ transplant recipients. Methods: We prospectively measured anti-HLA IgE antibodies in a cohort of kidney (n=60), liver, heart and lung (n=15 each) transplant recipients before and within one-year after transplantation, employing a single-antigen bead assay for HLA class I and class II antigens. Functional activity of anti-HLA IgE antibodies was assessed by an in vitro mediator release assay. Antibodies of the IgG1-4 subclasses and Th1 and Th2 cytokines were measured in anti-HLA IgE positive patients. Results: Pre-existing anti-HLA IgE antibodies were detected in 10% of renal recipients (including 3.3% IgE-DSA) and in 4.4% of non-renal solid organ transplant recipients (heart, liver and lung cohort). Anti-HLA IgE occurred only in patients that were positive for anti-HLA IgG, and most IgE positive patients had had a previous transplant. Only a small fraction of patients developed de novo anti-HLA IgE antibodies (1.7% of kidney recipients and 4.4% of non-renal recipients), whereas no de novo IgE-DSA was detected. IgG subclass antibodies showed a distinct pattern in patients who were positive for anti-HLA IgE. Moreover, patients with anti-HLA IgE showed elevated Th2 and also Th1 cytokine levels. Serum from IgE positive recipients led to degranulation of basophils in vitro, demonstrating functionality of anti-HLA IgE. Discussion: These data demonstrate that anti-HLA IgE antibodies occur at low frequency in kidney, liver, heart and lung transplant recipients. Anti-HLA IgE development is associated with sensitization at the IgG level, in particular through previous transplants and distinct IgG subclasses. Taken together, HLA specific IgE sensitization is a new phenomenon in solid organ transplant recipients whose potential relevance for allograft injury requires further investigation.

Lower magnitude and faster waning of antibody responses to SARS-CoV-2 vaccination in anti-TNF-α-treated IBD patients are linked to lack of activation and expansion of cTfh1 cells and impaired B memory cell formation.

BACKGROUND: Patients with inflammatory bowel disease (IBD) and healthy controls received primary SARS-CoV-2-mRNA vaccination and a booster after six months. Anti-TNF-α-treated patients showed significantly lower antibody (Ab) levels and faster waning than α4ß7-integrin-antagonist recipients and controls. This prospective cohort study aimed to elucidate the underlying mechanisms on the basis of circulating T-follicular helper cells (cTfh) and B memory cells. METHODS: We measured SARS-CoV-2- Wuhan and Omicron specific Abs, B- and T-cell subsets at baseline and kinetics of Spike (S)-specific B memory cells along with distributions of activated cTfh subsets before and after primary and booster vaccination. FINDINGS: Lower and faster waning of Ab levels in anti-TNF-α treated IBD patients was associated with low numbers of total and naïve B cells vs. expanded plasmablasts prior to vaccination. Along with their low Ab levels against Wuhan and Omicron VOCs, reduced S-specific B memory cells were identified after the 2nd dose which declined to non-detectable after 6 months. In contrast, IBD patients with α4ß7-integrin-antagonists and controls mounted and retained high Ab levels after the 2nd dose, which was associated with a pronounced increase in S-specific B memory cells that were maintained or expanded up to 6 months. Booster vaccination led to a strong increase of Abs with neutralizing capacity and S-specific B memory cells in these groups, which was not the case in anti-TNF-α treated IBD patients. Of note, Ab levels and S-specific B memory cells in particular post-booster correlated with the activation of cTfh1 cells after primary vaccination. INTERPRETATIONS: The reduced magnitude, persistence and neutralization capacity of SARS-CoV-2 specific Abs after vaccination in anti-TNF-α-treated IBD patients were associated with impaired formation and maintenance of S-specific B memory cells, likely due to absent cTfh1 activation leading to extra-follicular immune responses and diminished B memory cell diversification. These observations have implications for patient-tailored vaccination schedules/vaccines in anti-TNF-α-treated patients, irrespective of their underlying disease. FUNDING: The study was funded by third party funding of the Institute of Specific Prophylaxis and Tropical Medicine at the Medical University Vienna. The funders had no role in study design, data collection, data analyses, interpretation, or writing of report.

Identification of a Synthetic Polyhydroxyphenolic Resveratrol Analogue, 3,3',4,4',5,5'-Hexahydroxy-trans-Stilbene with Anti-SARS-CoV-2 Activity.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has been causing the COVID-19 pandemic since December 2019, with over 600 million infected persons worldwide and over six million deaths. We investigated the anti-viral effects of polyphenolic green tea ingredients and the synthetic resveratrol analogue 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (HHS), a compound with antioxidant, antitumor and anti-HIV properties. In the TCID50 assay, four out of nine green tea constituents showed minor to modest cell protective effects, whereas HHS demonstrated the highest reduction (1103-fold) of the TCID50, indicating pronounced inhibition of virus replication. HHS was also a highly effective inhibitor of SARS-CoV-2 proliferation in VeroE6 cells with an IC50 value of 31.1 µM. HSS also inhibited the binding of the receptor-binding domain (RBD) of the spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor (RBD-ACE2) binding with 29% at 100 µM and with 9.2% at 50 µM indicating that the SARS-CoV-2 inhibitory effect might at least in part be attributed to the inhibition of virus binding to ACE2. Based on the chemical similarity to other polyphenols, the oral bioavailability of HHS is likely also very low, resulting in blood levels far below the inhibitory concentration of EGCG against SARS-CoV-2 observed in vitro. However, administration of HHS topically as a nose or throat spray would increase concentrations several-fold above the minimal inhibitory concentration (MIC) in the mucosa and might reduce virus load when administered soon after infection. Due to these promising tissue culture results, further preclinical and clinical studies are warranted to develop HHS as an additional treatment option for SARS-CoV-2 infection to complement vaccines, which is and will be the main pillar to combat the COVID-19 pandemic.

Dissection of Antibody Responses of Gam-COVID-Vac-Vaccinated Subjects Suggests Involvement of Epitopes Outside RBD in SARS-CoV-2 Neutralization.

Millions of people have been vaccinated with Gam-COVID-Vac but fine specificities of induced antibodies have not been fully studied. Plasma from 12 naïve and 10 coronavirus disease 2019 (COVID-19) convalescent subjects was obtained before and after two immunizations with Gam-COVID-Vac. Antibody reactivity in the plasma samples (n = 44) was studied on a panel of micro-arrayed recombinant folded and unfolded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and 46 peptides spanning the spike protein (S) and by immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). The ability of Gam-COVID-Vac-induced antibodies to inhibit binding of the receptor-binding domain (RBD) to its receptor angiotensin converting enzyme 2 (ACE2) was investigated in a molecular interaction assay (MIA). The virus-neutralizing capacity of antibodies was studied by the pseudo-typed virus neutralization test (pVNT) for Wuhan-Hu-1 and Omicron. We found that Gam-COVID-Vac vaccination induced significant increases of IgG1 but not of other IgG subclasses against folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), and RBD in a comparable manner in naïve and convalescent subjects. Virus neutralization was highly correlated with vaccination-induced antibodies specific for folded RBD and a novel peptide (i.e., peptide 12). Peptide 12 was located close to RBD in the N-terminal part of S1 and may potentially be involved in the transition of the pre- to post-fusion conformation of the spike protein. In summary, Gam-COVID-Vac vaccination induced S-specific IgG1 antibodies in naive and convalescent subjects in a comparable manner. Besides the antibodies specific for RBD, the antibodies induced against a peptide close to the N-terminus of RBD were also associated with virus-neutralization.

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies.

More than three years ago, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused the unforeseen COVID-19 pandemic with millions of deaths. In the meantime, SARS-CoV-2 has become endemic and is now part of the repertoire of viruses causing seasonal severe respiratory infections. Due to several factors, among them the development of SARS-CoV-2 immunity through natural infection, vaccination and the current dominance of seemingly less pathogenic strains belonging to the omicron lineage, the COVID-19 situation has stabilized. However, several challenges remain and the possible new occurrence of highly pathogenic variants remains a threat. Here we review the development, features and importance of assays measuring SARS-CoV-2 neutralizing antibodies (NAbs). In particular we focus on in vitro infection assays and molecular interaction assays studying the binding of the receptor binding domain (RBD) with its cognate cellular receptor ACE2. These assays, but not the measurement of SARS-CoV-2-specific antibodies per se, can inform us of whether antibodies produced by convalescent or vaccinated subjects may protect against the infection and thus have the potential to predict the risk of becoming newly infected. This information is extremely important given the fact that a considerable number of subjects, in particular vulnerable persons, respond poorly to the vaccination with the production of neutralizing antibodies. Furthermore, these assays allow to determine and evaluate the virus-neutralizing capacity of antibodies induced by vaccines and administration of plasma-, immunoglobulin preparations, monoclonal antibodies, ACE2 variants or synthetic compounds to be used for therapy of COVID-19 and assist in the preclinical evaluation of vaccines. Both types of assays can be relatively quickly adapted to newly emerging virus variants to inform us about the magnitude of cross-neutralization, which may even allow us to estimate the risk of becoming infected by newly appearing virus variants. Given the paramount importance of the infection and interaction assays we discuss their specific features, possible advantages and disadvantages, technical aspects and not yet fully resolved issues, such as cut-off levels predicting the degree of in vivo protection.

Inhibition of SARS-CoV-2 Omicron BA.1 and BA.4 Variants After Fourth Vaccination or Tixagevimab and Cilgavimab Administration in Patients With Cancer.

This cohort study assesses the capacity of passive immunization and tixagevimab and cilgavimab to inhibit interaction between receptor-binding domains and angiotensin-converting enzyme 2 in patients with hemato-oncologic diseases.

Complex IgE sensitization patterns in ragweed allergic patients: Implications for diagnosis and specific immunotherapy.

Background: Ragweed (Ambrosia artemisiifolia) is one of the most important allergen sources, worldwide, causing severe respiratory allergic reactions in late summer and fall, in sensitized patients. Amb a 1 has been considered as the most important allergen in ragweed but 12 ragweed pollen allergens are known. The aim of our study was to investigate IgE reactivity profiles of ragweed allergic patients and to associate them with clinical symptoms. Methods: IgE sensitization profiles from clinically well-characterized ragweed allergic patients (n = 150) were analyzed using immunoblotted ragweed pollen extract. Immunoblot inhibition experiments were performed with two Amb a 1 isoforms and CCD markers and basophil activation experiments were performed with IgE serum before and after depletion of Amb a 1-specific IgE. Results: By IgE-immunoblotting 19 different IgE reactivity patterns with and without Amb a 1-sensitization were found. The majority of patients (>95%) suffered from rhino-conjunctivitis, around 60% reported asthma-like symptoms and about 25% had skin reactions. Patients with complex IgE sensitization profiles tended to have more clinical symptoms. Serum with and without Amb a 1-specific IgE induced basophil activation. Conclusions: Ragweed pollen allergic patients exhibit complex IgE reactivity profiles to ragweed allergens including Amb a 1 isoforms and cross-reactive carbohydrates indicating the importance of Amb a 1 isoforms and additional allergens for diagnosis and allergen-specific immunotherapy of ragweed allergy.

Combined assessment of S- and N-specific IL-2 and IL-13 secretion and CD69 neo-expression for discrimination of post-infection and post-vaccination cellular SARS-CoV-2-specific immune response.

BACKGROUND: Antibody-based tests are available for measuring SARS-CoV-2-specific immune responses but fast T-cell assays remain scarce. Robust T cell-based tests are needed to differentiate specific cellular immune responses after infection from those after vaccination. METHODS: One hundred seventeen individuals (COVID-19 convalescent patients: n = 40; SARS-CoV-2 vaccinees: n = 41; healthy controls: n = 36) were evaluated for SARS-CoV-2-specific cellular immune responses (proliferation, Th1, Th2, Th17, and inflammatory cytokines, activation-induced marker [AIM] expression) by incubating purified peripheral blood mononuclear cells (PBMC) or whole blood (WB) with SARS-CoV-2 peptides (S, N, or M), vaccine antigens (tetanus toxoid, tick borne encephalitis virus) or polyclonal stimuli (Staphylococcal enterotoxin, phytohemagglutinin). RESULTS: N-peptide mix stimulation of WB identified the combination of IL-2 and IL-13 secretion as superior to IFN-γ secretion to discriminate between COVID-19-convalescent patients and healthy controls (p < .0001). Comparable results were obtained with M- or S-peptides, the latter almost comparably recalled IL-2, IFN-γ, and IL-13 responses in WB of vaccinees. Analysis 10 months as opposed to 10 weeks after COVID-19, but not allergic disease status, positively correlated with IL-13 recall responses. WB cytokine responses correlated with cytokine and proliferation responses of PBMC. Antigen-induced neo-expression of the C-type lectin CD69 on CD4+ (p < .0001) and CD8+ (p = .0002) T cells informed best about the SARS-CoV-2 exposure status with additional benefit coming from CD25 upregulation. CONCLUSION: Along with N- and S-peptide-induced IL-2 and CD69 neo-expression, we suggest to include the type 2 cytokine IL-13 as T-cellular recall marker for SARS-CoV-2 specific T-cellular immune responses after infection and vaccination.

Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

Vaccine based on folded receptor binding domain-PreS fusion protein with potential to induce sterilizing immunity to SARS-CoV-2 variants.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global COVID-19 pandemic. One possibility to control the pandemic is to induce sterilizing immunity through the induction and maintenance of neutralizing antibodies preventing SARS-CoV-2 from entering human cells to replicate in. METHODS: We report the construction and in vitro and in vivo characterization of a SARS-CoV-2 subunit vaccine (PreS-RBD) based on a structurally folded recombinant fusion protein consisting of two SARS-CoV-2 Spike protein receptor-binding domains (RBD) fused to the N- and C-terminus of hepatitis B virus (HBV) surface antigen PreS to enable the two unrelated proteins serving as immunologic carriers for each other. RESULTS: PreS-RBD, but not RBD alone, induced a robust and uniform RBD-specific IgG response in rabbits. Currently available genetic SARS-CoV-2 vaccines induce mainly transient IgG1 responses in vaccinated subjects whereas the PreS-RBD vaccine induced RBD-specific IgG antibodies consisting of an early IgG1 and sustained IgG4 antibody response in a SARS-CoV-2 naive subject. PreS-RBD-specific IgG antibodies were detected in serum and mucosal secretions, reacted with SARS-CoV-2 variants, including the omicron variant of concern and the HBV receptor-binding sites on PreS of currently known HBV genotypes. PreS-RBD-specific antibodies of the immunized subject more potently inhibited the interaction of RBD with its human receptor ACE2 and their virus-neutralizing titers (VNTs) were higher than median VNTs in a random sample of healthy subjects fully immunized with registered SARS-CoV-2 vaccines or in COVID-19 convalescent subjects. CONCLUSION: The PreS-RBD vaccine has the potential to serve as a combination vaccine for inducing sterilizing immunity against SARS-CoV-2 and HBV by stopping viral replication through the inhibition of cellular virus entry.

Lack of Induction of RBD-Specific Neutralizing Antibodies despite Repeated Heterologous SARS-CoV-2 Vaccination Leading to Seroconversion and Establishment of T Cell-Specific Memory in a Patient in Remission of Multiple Myeloma.

BACKGROUND: Prophylactic vaccination against infectious diseases may induce a state of long-term protection in the otherwise healthy host. However, the situation is less predictable in immunocompromised patients and may require adjustment of vaccination schedules and/or basic therapy. METHODS: A patient in full remission of multiple myeloma since the last three years and on long-term maintenance therapy with pomalidomide, a drug inhibiting angiogenesis and myeloma cell growth, was vaccinated twice with Comirnaty followed by two vaccinations with Vaxzevria. Seroconversion and SARS-CoV-2-specific cellular responses were monitored. RESULTS: No signs of seroconversion or T cellular memory were observed after the first "full immunization" with Comirnaty. Consequently, long-term-maintenance therapy with Pomalidomide was stopped and two additional shots of Vaxzevria were administered after which the patient seroconverted with Spike(S)-protein specific antibody levels reaching 49 BAU/mL, mild S-peptide pool-specific T cell proliferation, effector cytokine production (IL-2, IL-13), and T cellular activation with increased numbers of CD3+CD4+CD25+ T cells as compared to vaccinated and non-vaccinated control subjects. However, despite suspension of immunosuppression and administration of in total four consecutive heterologous SARS-CoV-2 vaccine shots, the patient did not develop neutralizing RBD-specific antibodies. CONCLUSIONS: Despite immunomonitoring-based adjustment of vaccination and/or therapy schedules vaccination success, with clear correlates of protection, the development of RBD-specific antibodies could not be achieved in the immunocompromised patient with current SARS-CoV-2 vaccines. Thus, our report emphasizes the need for improved active and passive immunization strategies for SARS-CoV-2 infections.

Characterization of the antibody response to SARS-CoV-2 in a mildly affected pediatric population.

BACKGROUND: While children usually experience a mild course of COVID-19, and a severe disease is more common in adults, the features, specificities, and functionality of the SARS-CoV-2-specific antibody response in the pediatric population are of interest. METHODS: We performed a detailed analysis of IgG antibodies specific for SARS-CoV-2-derived antigens S and RBD by ELISA in 26 SARS-CoV-2 seropositive schoolchildren with mild or asymptomatic disease course, and in an equally sized, age- and gender-matched control group. Furthermore, a detailed mapping of IgG reactivity to a panel of microarrayed SARS-CoV-2 proteins and S-derived peptides was performed by microarray technology. The capacity of the antibody response to block RBD-ACE2 binding and virus neutralization were assessed. Results were compared with those obtained in an adult COVID-19 convalescent population. RESULTS: After mild COVID-19, anti-S and RBD-specific IgG antibodies were developed by 100% and 84.6% of pediatric subjects, respectively. No difference was observed in regards to symptoms and gender. Mounted antibodies recognized conformational epitopes of the spike protein and were capable to neutralize the virus up to a titer of ≥80 and to inhibit the ACE2-RBD interaction by up to 65%. SARS-CoV-2-specific IgG responses in children were comparable to mildly affected adult patients. CONCLUSION: SARS-CoV-2 asymptomatic and mildly affected pediatric patients develop a SARS-CoV-2-specific antibody response, which is comparable regarding antigen, epitope recognition, and the ability to inhibit the RBD-ACE2 interaction to that observed in adult patients after mild COVID-19.

Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes.

BACKGROUND: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. METHODS: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus. RESULTS: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1 , and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. CONCLUSION: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2-neutralizing antibodies conferring sterilizing immunity.

Multiprofessional perinatal care in a pregnant patient with acute respiratory distress syndrome due to COVID-19.

BACKGROUND: The coronavirus disease (COVID-19) pandemic has caused ongoing challenges in health services worldwide. Despite the growing body of literature on COVID-19, reports on perinatal care in COVID-19 cases are limited. CASE PRESENTATION: We describe a case of severe acute respiratory distress syndrome (ARDS) in a 36-year-old G5/P2 pregnant woman with morbid obesity, confirmed severe acute respiratory syndrome coronavirus 2 infection, and fulminant respiratory failure. At 28+ 1 gestational weeks, the patient delivered an uninfected newborn. Using ImmunoCAP ISAC® technology, we found no immunoglobulin (Ig) M antibodies, suggesting that no mother-to-child viral transmission occurred during pregnancy or delivery. The maternal respiratory state improved rapidly after delivery; both maternal and neonatal outcomes were encouraging given the early gestational age and fulminant course of respiratory failure in our patient. CONCLUSIONS: The management of ARDS in pregnant women with COVID-19 is complex and requires an individualized, multidisciplinary approach, while considering maternal and fetal outcomes.