Differential decline of SARS-CoV-2-specific antibody levels, innate and adaptive immune cells, and shift of Th1/inflammatory to Th2 serum cytokine levels long after first COVID-19.

BACKGROUND: SARS-CoV-2 has triggered a pandemic and contributes to long-lasting morbidity. Several studies have investigated immediate cellular and humoral immune responses during acute infection. However, little is known about long-term effects of COVID-19 on the immune system. METHODS: We performed a longitudinal investigation of cellular and humoral immune parameters in 106 non-vaccinated subjects ten weeks (10 w) and ten months (10 m) after their first SARS-CoV-2 infection. Peripheral blood immune cells were analyzed by multiparametric flow cytometry, serum cytokines were examined by multiplex technology. Antibodies specific for the Spike protein (S), the receptor-binding domain (RBD) and the nucleocapsid protein (NC) were determined. All parameters measured 10 w and 10 m after infection were compared with those of a matched, noninfected control group (n = 98). RESULTS: Whole blood flow cytometric analyses revealed that 10 m after COVID-19, convalescent patients compared to controls had reduced absolute granulocyte, monocyte, and lymphocyte counts, involving T, B, and NK cells, in particular CD3+CD45RA+CD62L+CD31+ recent thymic emigrant T cells and non-class-switched CD19+IgD+CD27+ memory B cells. Cellular changes were associated with a reversal from Th1- to Th2-dominated serum cytokine patterns. Strong declines of NC- and S-specific antibody levels were associated with younger age (by 10.3 years, p < .01) and fewer CD3-CD56+ NK and CD19+CD27+ B memory cells. Changes of T-cell subsets at 10 m such as normalization of effector and Treg numbers, decline of RTE, and increase of central memory T cell numbers were independent of antibody decline pattern. CONCLUSIONS: COVID-19 causes long-term reduction of innate and adaptive immune cells which is associated with a Th2 serum cytokine profile. This may provide an immunological mechanism for long-term sequelae after COVID-19.

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.

Determinants of immunoglobulin G responses to respiratory syncytial virus and rhinovirus in children and adults.

Introduction: Exposure to respiratory viruses is a significant cause of morbidity and affects virus-specific antibody levels. Little is known about determinants associated with immune response to these viruses. We aimed to investigate the determinants of respiratory syncytial virus (RSV)- and rhinovirus (RV)- specific IgG responses in both children and adults. Methods: The study is based on the EGEA cohort, composed of 530 samples of children in EGEA1 (1991-95) and 1241 samples of adults in EGEA2 (2003-07). Cumulative RV-specific IgG levels (species A, B and C) and IgG levels to RSV-G protein were measured by using micro-array technoloy. Multiple linear mixed models (random effect to account for familial dependence) were performed to assess associations between age, sex, body mass index (BMI), tobacco smoke exposure and season of blood sampling with RSV-and RV-specific IgG levels. Results: In children (11.1 ± 2.8 years old, 57% boys), higher RV-specific IgG levels were associated with older age (only for RV-B), female sex and lower BMI, while only older age was associated with higher RSV-specific IgG levels. In adults (43.5 ± 16.7 years old, 48% men), younger age, female sex, lower BMI, active smoking and all seasons except summer were associated with higher RV-specific IgG levels. Older age, active smoking and all seasons except summer were associated with higher RSV-specific IgG levels. Conclusion: Personal and seasonal determinants of RSV- and RV-specific IgG levels seem to vary according to the respiratory virus type and between children and adults, suggesting different patterns of responses along the life course.

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.

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.

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.

Dissociation of the respiratory syncytial virus F protein-specific human IgG, IgA and IgM response.

Human respiratory syncytial virus (RSV) is one of the most important causes of severe respiratory tract infections in early childhood. The only prophylactic protection is the neutralizing antibody, palivizumab, which targets a conformational epitope of the RSV fusion (F) protein. The F protein is generated as a F0 precursor containing two furin cleavage sites allowing excision of the P27 fragment and then gives rise to a fusion-competent version consisting of the N-terminal F2 subunit and the a C-terminal F1 subunits linked by two disulphide bonds. To investigate natural human F-specific antibody responses, F2 conferring the species-specificity of RSV, was expressed in Escherichia coli. Furthermore, the F0 protein, comprising both subunits F2 and F1, was expressed as palivizumab-reactive glycoprotein in baculovirus-infected insect cells. Six overlapping F2-derived peptides lacking secondary structure were synthesized. The analysis of IgG, IgA and IgM responses of adult subjects to native versions and denatured forms of F2 and F0 and to unfolded F2-derived peptides revealed that mainly non-conformational F epitopes, some of which represented cryptic epitopes which are not exposed on the proteins were recognized. Furthermore, we found a dissociation of IgG, IgA and IgM antibody responses to F epitopes with F2 being a major target for the F-specific IgM response. The scattered and dissociated immune response to F may explain why the natural RSV-specific antibody response is only partially protective underlining the need for vaccines focusing human antibody responses towards neutralizing RSV epitopes.

Immunological imprint of COVID-19 on human peripheral blood leukocyte populations.

BACKGROUND: SARS-CoV-2 has triggered a pandemic that is now claiming many lives. Several studies have investigated cellular immune responses in COVID-19-infected patients during disease but little is known regarding a possible protracted impact of COVID-19 on the adaptive and innate immune system in COVID-19 convalescent patients. METHODS: We used multiparametric flow cytometry to analyze whole peripheral blood samples and determined SARS-CoV-2-specific antibody levels against the S-protein, its RBD-subunit, and viral nucleocapsid in a cohort of COVID-19 convalescent patients who had mild disease ~10 weeks after infection (n = 109) and healthy control subjects (n = 98). Furthermore, we correlated immunological changes with clinical and demographic parameters. RESULTS: Even ten weeks after disease COVID-19 convalescent patients had fewer neutrophils, while their cytotoxic CD8+ T cells were activated, reflected as higher HLA-DR and CD38 expression. Multiparametric regression analyses showed that in COVID-19-infected patients both CD3+ CD4+ and CD3+ CD8+ effector memory cells were higher, while CD25+ Foxp3+ T regulatory cells were lower. In addition, both transitional B cell and plasmablast levels were significantly elevated in COVID-19-infected patients. Fever (duration, level) correlated with numbers of central memory CD4+ T cells and anti-S and anti-RBD, but not anti-NC antibody levels. Moreover, a "young immunological age" as determined by numbers of CD3+ CD45RA+ CD62L+ CD31+ recent thymic emigrants was associated with a loss of sense of taste and/or smell. CONCLUSION: Acute SARS-CoV-2 infection leaves protracted beneficial (ie, activation of T cells) and potentially harmful (ie, reduction of neutrophils) imprints in the cellular immune system in addition to induction of specific antibody responses.

Features of the Human Antibody Response against the Respiratory Syncytial Virus Surface Glycoprotein G.

Respiratory syncytial virus (RSV) infections are a major cause of serious respiratory disease in infants. RSV occurs as two major subgroups A and B, which mainly differ regarding the surface glycoprotein G. The G protein is important for virus attachment and G-specific antibodies can protect against infection. We expressed the surface-exposed part of A2 strain-derived G (A2-G) in baculovirus-infected insect cells and synthesized overlapping peptides spanning complete A2-G. The investigation of the natural IgG response of adult subjects during a period of one year showed that IgG antibodies (i) recognize G significantly stronger than the fusion protein F0, (ii) target mainly non-conformational, sequential peptide epitopes from the exposed conserved region but also buried peptides, and (iii) exhibit a scattered but constant recognition profile during the observation period. The IgG subclass reactivity profile (IgG1 > IgG2 > IgG4 = IgG3) was indicative of a mixed Th1/Th2 response. Two strongly RSV-neutralizing sera including the 1st WHO standard contained high IgG anti-G levels. G-specific IgG increased strongly in children after wheezing attacks suggesting RSV as trigger factor. Our study shows that RSV G and G-derived peptides are useful for serological diagnosis of RSV-triggered exacerbations of respiratory diseases and underlines the importance of G for development of RSV-neutralizing vaccines.

Toward personalization of asthma treatment according to trigger factors.

Asthma is a severe and chronic disabling disease affecting more than 300 million people worldwide. Although in the past few drugs for the treatment of asthma were available, new treatment options are currently emerging, which appear to be highly effective in certain subgroups of patients. Accordingly, there is a need for biomarkers that allow selection of patients for refined and personalized treatment strategies. Recently, serological chip tests based on microarrayed allergen molecules and peptides derived from the most common rhinovirus strains have been developed, which may discriminate 2 of the most common forms of asthma, that is, allergen- and virus-triggered asthma. In this perspective, we argue that classification of patients with asthma according to these common trigger factors may open new possibilities for personalized management of asthma.