Impact of Omicron Variant Infection on Assessment of Spike-Specific Immune Responses Using the EUROIMMUN Quan-T-Cell SARS-CoV-2 Assay and Roche Elecsys Anti-SARS-CoV-2-S.

The currently prevailing variants of SARS-CoV-2 are subvariants of the Omicron variant. The aim of this study was to analyze the effect of mutations in the Spike protein of Omicron on the results Quan-T-Cell SARS-CoV-2 assays and Roche Elecsys anti-SARS-CoV-2 anti-S1. Omicron infected subjects ((n = 37), vaccinated (n = 20) and unvaccinated (n = 17)) were recruited approximately 3 weeks after a positive PCR test. The Quan-T-Cell SARS-CoV-2 assays (EUROIMMUN) using Wuhan and the Omicron adapted antigen assay and a serological test (Roche Elecsys anti-SARS-CoV-2 anti-S1) were performed. Using the original Wuhan SARS-CoV-2 IGRA TUBE, in 19 of 21 tested Omicron infected subjects, a positive IFNy response was detected, while 2 non-vaccinated but infected subjects did not respond. The Omicron adapted antigen tube resulted in comparable results. In contrast, the serological assay detected a factor 100-fold lower median Spike-specific RBD antibody concentration in non-vaccinated Omicron infected patients (n = 12) compared to patients from the pre Omicron era (n = 12) at matched time points, and eight individuals remained below the detection threshold for positivity. For vaccinated subjects, the Roche assay detected antibodies in all subjects and showed a 400 times higher median specific antibody concentration compared to non-vaccinated infected subjects in the pre-Omicron era. Our results suggest that Omicron antigen adapted IGRA stimulator tubes did not improve detection of SARS-CoV-2-specific T-cell responses in the Quant-T-Cell-SARS-CoV-2 assay. In non-vaccinated Omicron infected individuals, the Wuhan based Elecsys anti-SARS-CoV-2 anti-S1 serological assay results in many negative results at 3 weeks after diagnosis.

High Incidence of SARS-CoV-2 Variant of Concern Breakthrough Infections Despite Residual Humoral and Cellular Immunity Induced by BNT162b2 Vaccination in Healthcare Workers: A Long-Term Follow-Up Study in Belgium.

To mitigate the massive COVID-19 burden caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccination campaigns were initiated. We performed a single-center observational trial to monitor the mid- (3 months) and long-term (10 months) adaptive immune response and to document breakthrough infections (BTI) in healthcare workers (n = 84) upon BNT162b2 vaccination in a real-world setting. Firstly, serology was determined through immunoassays. Secondly, antibody functionality was analyzed via in vitro binding inhibition and pseudovirus neutralization and circulating receptor-binding domain (RBD)-specific B cells were assessed. Moreover, the induction of SARS-CoV-2-specific T cells was investigated by an interferon-γ release assay combined with flowcytometric profiling of activated CD4+ and CD8+ T cells. Within individuals that did not experience BTI (n = 62), vaccine-induced humoral and cellular immune responses were not correlated. Interestingly, waning over time was more pronounced within humoral compared to cellular immunity. In particular, 45 of these 62 subjects no longer displayed functional neutralization against the delta variant of concern (VoC) at long-term follow-up. Noteworthily, we reported a high incidence of symptomatic BTI cases (17.11%) caused by alpha and delta VoCs, although vaccine-induced immunity was only slightly reduced compared to subjects without BTI at mid-term follow-up.

Differences in Immunogenicity of Three Different Homo- and Heterologous Vaccination Regimens against SARS-CoV-2.

Background: Due to findings on adverse reactions and clinical efficacy of different vaccinations against SARS-CoV-2, the administration of vaccination regimens containing both adenoviral vector vaccines and mRNA-based vaccines has become common. Data are still needed on the direct comparison of immunogenicity for these different regimens. Methods: We compared markers for immunogenicity (anti-S1 IgG/IgA, neutralizing antibodies, and T-cell response) with three different vaccination regimens (homologous ChAdOx1 nCoV-19 (n = 103), or mixture of ChAdOx1 nCoV-19 with mRNA-1273 (n = 116) or BNT162b2 (n = 105)) at two time points: the day of the second vaccination as a baseline and 14 days later. Results: All examined vaccination regimens elicited measurable immune responses that were significantly enhanced after the second dose. Homologous ChAdOx1 nCoV-19 was markedly inferior in immunogenicity to all other examined regimens after administration of the second dose. Between the heterologous regimens, mRNA-1273 as second dose induced greater antibody responses than BNT162b2, with no difference found for neutralizing antibodies and T-cell response. Discussion: While these findings allow no prediction about clinical protection, from an immunological point of view, vaccination against SARS-CoV-2 with an mRNA-based vaccine at one or both time points appears preferable to homologous vaccination with ChAdOx1 nCoV-19. Whether or not the demonstrated differences between the heterologous regimens are of clinical significance will be subject to further research.

B-cell responses to vaccination with BNT162b2 and mRNA-1273 6 months after second dose.

OBJECTIVES: To examine the state of B-cell immunity 6 months after the second vaccination against SARS-CoV-2 in comparison to the state observed 2 weeks after vaccination. METHODS: Sera of 439 participants, whose immune responses to two doses of an mRNA-based vaccine (BNT162b2 or mRNA-1273) were previously characterized, was examined for anti-S1 IgG and IgA, anti-NCP IgG and neutralizing antibodies (nAb), and antinuclear antibodies (ANA). RESULTS: Levels of all examined markers decreased significantly from 2 weeks to 6 months after second vaccination (anti-S1 IgG: 3744 ± 2571.4 vs. 253 ± 144 binding antibody units (BAU)/mL; anti-S1 IgA: 12 ± 0 vs. 1.98 ± 1.75 optical density (OD) ratio; nAb: 100% ± 0% vs. 82% ± 19.3%), the vast majority of participants retaining reactive levels of anti-S1 IgG (436/439) and anti-S1 IgA (334/439) at 6 months. Immune responses were stronger for mRNA-1273 compared with BNT162b2 (anti-S1 IgG: 429 ± 289 vs. 243 ± 143 BAU/mL; anti-S1 IgA: 5.38 ± 3.91 vs. 1.89 ± 1.53 OD ratio; nAb: 90.5% ± 12.6% vs. 81% ± 19.3%). There was no meaningful influence of sex and age on the examined markers. There was a strong correlation between anti-S1 IgG and the surrogate neutralization assay (rho = 0.91, p <0.0001), but not for for IgA and the surrogate neutralization assay (rho = 0.52, p <0.0001). There was a ceiling effect for the association between anti-S1 IgG titres and the inhibition of binding between S1 and ACE2. ANA prevalence was unchanged from 2 weeks to 6 months after the second vaccination (87/498 vs. 77/435), as were the median ANA titres (1:160 vs. 1:160). DISCUSSION: Although the clinical consequences of decreasing anti-SARS-CoV-2 antibody titres cannot be estimated with certainty, a lowered degree of clinical protection against SARS-CoV-2 is possible. Persistently stronger responses to mRNA-1273 suggest that it might confer greater protection than BNT162b2, even 6 months after the second vaccination. Neither examined vaccinations induced ANA within the examined time frame.

Kinetics of the Antibody Response to Boostering With Three Different Vaccines Against SARS-CoV-2.

BACKGROUND: Heterologous vaccinations against SARS-CoV-2 with ChAdOx1 nCoV-19 and a second dose of an mRNA-based vaccine have been shown to be more immunogenic than homologous ChAdOx1 nCoV-19. In the current study, we examined the kinetics of the antibody response to the second dose of three different vaccination regimens (homologous ChAdOx1 nCoV-19 vs. ChAdOx1 nCoV-19 + BNT162b2 or mRNA-1273) against SARS-CoV-2 in a longitudinal manner; whether there are differences in latency or amplitude of the early response and which markers are most suitable to detect these responses. METHODS: We performed assays for anti-S1 IgG and IgA, anti-NCP IgG and a surrogate neutralization assay on serum samples collected from 57 participants on the day of the second vaccination as well as the following seven days. RESULTS: All examined vaccination regimens induced detectable antibody responses within the examined time frame. Both heterologous regimens induced responses earlier and with a higher amplitude than homologous ChAdOx1 nCoV-19. Between the heterologous regimens, amplitudes were somewhat higher for ChAdOx1 nCoV-19 + mRNA-1273. There was no difference in latency between the IgG and IgA responses. Increases in the surrogate neutralization assay were the first changes to be detectable for all regimens and the only significant change seen for homologous ChAdOx1 nCoV-19. DISCUSSION: Both examined heterologous vaccination regimens are superior in immunogenicity, including the latency of the response, to homologous ChAdOx1 nCoV-19. While the IgA response has a shorter latency than the IgG response after the first dose, no such difference was found after the second dose, implying that both responses are driven by separate plasma cell populations. Early and steep increases in surrogate neutralization levels suggest that this might be a more sensitive marker for antibody responses after vaccination against SARS-CoV-2 than absolute levels of anti-S1 IgG.

The temporal course of T- and B-cell responses to vaccination with BNT162b2 and mRNA-1273.

OBJECTIVES: To investigate the response of the immune system (and its influencing factors) to vaccination with BNT162b2 or mRNA-1273. METHODS: 531 vaccinees, recruited from healthcare professionals, donated samples before, in between, and after the administration of the two doses of the vaccine. T- and B-cell responses were examined via interferon-γ (IFN-γ) release assay, and antibodies against different epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (S1 and NCP) were detected via ELISA and surrogate neutralization assay. Results were correlated with influencing factors such as age, sex, prior infection, vaccine received (BNT162b2 or mRNA-1273), and immunosuppression. Furthermore, antinuclear antibodies (ANAs) were measured to screen for autoimmune responses following vaccination with an mRNA vaccine. RESULTS: No markers of immunity against SARS-CoV-2 were found before the first vaccination. Two weeks after it, specific responses against SARS-CoV-2 were already measurable (median ± median absolute deviation (MAD): anti-S1 IgG 195.5 ± 172.7 BAU/mL; IgA 6.7 ± 4.9 OD; surrogate neutralization 39 ± 23.7%), and were significantly increased two weeks after the second dose (anti-S1 IgG 3744 ± 2571.4 BAU/mL; IgA 12 ± 0 OD; surrogate neutralization 100 ± 0%, IFN-γ 1897.2 ± 886.7 mIU/mL). Responses were stronger for younger participants (this difference decreasing after the second dose). Further influences were previous infection with SARS-CoV-2 (causing significantly stronger responses after the first dose compared to unexposed individuals (p ≤ 0.0001)) and the vaccine received (significantly stronger reactions for recipients of mRNA-1273 after both doses, p < 0.05-0.0001). Some forms of immunosuppression significantly impeded the immune response to the vaccination (with no observable immune response in three immunosuppressed participants). There was no significant induction of ANAs by the vaccination (no change in qualitative ANA results (p 0.2592) nor ANA titres (p 0.08) from pre-to post-vaccination. CONCLUSIONS: Both vaccines elicit strong and specific immune responses against SARS-CoV-2 which become detectable one week (T-cell response) or two weeks (B-cell response) after the first dose.

Long-Term Course of Humoral and Cellular Immune Responses in Outpatients After SARS-CoV-2 Infection.

Characterization of the naturally acquired B and T cell immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for the development of public health and vaccination strategies to manage the burden of COVID-19 disease. We conducted a prospective, cross-sectional analysis in COVID-19 recovered patients at various time points over a 10-month period in order to investigate how circulating antibody levels and interferon-gamma (IFN-γ) release by peripheral blood cells change over time following natural infection. From March 2020 till January 2021, we enrolled 412 adults mostly with mild or moderate disease course. At each study visit, subjects donated peripheral blood for testing of anti-SARS-CoV-2 IgG antibodies and IFN-γ release after SARS-CoV-2 S-protein stimulation. Anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies were positive in 316 of 412 (76.7%) and borderline in 31 of 412 (7.5%) patients. Our confirmation assay for the presence of neutralizing antibodies was positive in 215 of 412 (52.2%) and borderline in 88 of 412 (21.4%) patients. Likewise, in 274 of 412 (66.5%) positive IFN-γ release and IgG antibodies were detected. With respect to time after infection, both IgG antibody levels and IFN-γ concentrations decreased by about half within 300 days. Statistically, production of IgG and IFN-γ were closely associated, but on an individual basis, we observed patients with high-antibody titres but low IFN-γ levels and vice versa. Our data suggest that immunological reaction is acquired in most individuals after natural infection with SARS-CoV-2 and is sustained in the majority of patients for at least 10 months after infection after a mild or moderate disease course. Since, so far, no robust marker for protection against COVID-19 exists, we recommend utilizing both, IgG and IFN-γ release for an individual assessment of the immunity status.

The HIF-1α/LC3-II Axis Impacts Fungal Immunity in Human Macrophages.

The fungal pathogen Histoplasma capsulatum causes a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity to H. capsulatum in mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced by H. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery of H. capsulatum from macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested that H. capsulatum exploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth of H. capsulatum Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.

Fc Receptor Inhibition Reduces Susceptibility to Oxidative Stress in Human RPE Cells Treated with Bevacizumab, but not Aflibercept.

BACKGROUND/AIMS: VEGF-A is induced by oxidative stress, and functions as a survival factor for various cell types, including retinal pigment epithelial (RPE) cells. Anti-vascular endothelial growth factor (VEGF) drugs like aflibercept and bevacizumab have shown to be most effective in treating neovascular age-related macular degeneration (AMD), however uptake of the drugs might lead to interference with cell physiology. Herein, we evaluated the significance of the Fc receptor (FcR) within this context and moreover explored the impact of VEGF inhibition under normal conditions as well as under oxidative stress, in terms of potential adverse effects. METHODS: ARPE-19 (human RPE) cells were treated with aflibercept and bevacizumab in presence or absence of H2O2 as oxidative stress stimulus. After 24h cells were evaluated for drug uptake, VEGF-A expression and secretion, levels of intracellular reactive oxygen species (ROS) as well as cell proliferation. Experiments were repeated with cells being pre-incubated with an FcR inhibitor prior to drug application. RESULTS: Both drugs inhibited extracellular levels of VEGF-A and were taken up into the RPE, resulting in significantly reduced intracellular levels of VEGF-A. When oxidative stress was applied, intracellular ROS levels in cells treated with both drugs rose, and cell proliferation was reduced. Prior incubation with the FcR inhibitor lessened the uptake of bevacizumab, but not aflibercept into RPE cells, and simultaneously enhanced cell survival under oxidative stress conditions. CONCLUSIONS: Our results indicate that uptake and accumulation of aflibercept and bevacizumab within RPE cells affect the intracellular VEGF-A metabolism negatively, leading to a biologically relevant reduced cell survival under oxidative stress. The FcR plays a substantial role in the uptake of bevacizumab, but not aflibercept, which allows an enhanced RPE cell survival through FcR blockage in an environment dominated by oxidative stress, as clinically significant for various inflammatory retinal disorders.