Search Results (364)

Despite the prevalence of fucosylated IgG in plasma, specific IgGs with low core fucosylation sporadically emerge in response to virus infections and blood cell alloantigens. This low fucosylation of IgG is implicated in the pathogenesis of SARS-CoV-2 and dengue infections. In COVID-19, the presence of IgGs with low core fucosylation (afucosylated IgGs) targeting spike protein predicts disease progression to a severe form and actively mediates this progression. This study reveals that SARS-CoV-2 infection of megakaryocytes promotes the generation of pathogenic afucosylated anti-spike IgGs, leading to outcomes, such as pulmonary vascular thrombosis, acute lung injury, and mortality in FcγRIIa-transgenic mice. Platelets from mice injected with virus-infected human megakaryocytes express significant activation biomarkers, indicating a direct link between the immune response and platelet activation. Mice injected with virus-infected human megakaryocytes demonstrate an elevated rate of thrombus formation induced by FeCl₃ (4%) and a reduction in bleeding time, emphasizing the intricate interplay of viral infection, immune response, and hemostatic complications. Treatment with inhibitors targeting FcγRIIa, serotonin, or complement anaphylatoxins of mice injected with spike-expressing MKs successfully prevents observed platelet activation, thrombus formation, and bleeding abnormalities, offering potential therapeutic strategies for managing severe outcomes associated with afucosylated IgGs in COVID-19 and related disorders. Full article

(1) Background: The ongoing global health threat posed by SARS-CoV-2 requires reliable and accessible diagnostic tools, especially in resource-limited settings where RT-qPCR may be impractical. This study describes the development and validation of two enzyme-linked immunosorbent assays (ELISA) designed to detect anti-SARS-CoV-2 IgG antibodies employing recombinant S1 and S2 spike protein subunits. (2) Methods: The assays were optimized and validated using serum samples from 354 RT-qPCR-confirmed hospitalized patients and 337 pre-pandemic blood donors. (3) Results: The S1-based ELISA achieved a 52.8% sensitivity and a specificity of 93.5%, with an area under the ROC curve (AUC) of 71.6%. In contrast, the S2-based ELISA demonstrated superior diagnostic performance, with a sensitivity of 63.7%, a specificity of 99.7%, and an AUC of 83.1%. Cross-reactivity analysis using sera from individuals with unrelated infectious diseases confirmed the high specificity of the S2-ELISA. Time-stratified analysis revealed that sensitivity increased with time, peaking between 15 and 21 days post-symptom onset. Compared to commercial serological assays, the S2-ELISA demonstrated comparable or improved performance, particularly in specificity and diagnostic odds ratio. (4) Conclusions: The S2-ELISA offers a robust, highly specific, and operationally simple tool for serological detection of SARS-CoV-2 infection. Its strong diagnostic performance and accessibility make it well-suited for implementation in diverse epidemiological settings, particularly where molecular testing is limited. The development of affordable, validated serological assays such as this is critical for strengthening surveillance, understanding transmission dynamics, and informing public health responses. Full article

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG_2a and IgG₁ after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity. Full article

Background/Objectives: Patients suffering from inflammatory bowel diseases (IBDs) undergoing treatment with anti-TNF antibodies mount a diminished humoral immune response to vaccination against SARS-CoV-2 compared to healthy controls. The characterization of variant-specific immune responses is particularly warranted among immunosuppressed patients, where reduced responses may necessitate further medical interventions. Methods: This pilot study investigated the humoral immune response of vaccinated IBD patients on anti-TNF medication and a comparable group of healthy individuals against the viral variants Alpha, Beta, Gamma, Delta, and Omicron BA.1 and BA.5. While total IgG antibodies targeting the receptor binding site of the spike protein of SARS-CoV-2 were quantified using a chemiluminescence microparticle immunoassay (CMIA), their potential neutralizing capacity was determined using commercial and variant-specific in-house surrogate virus neutralization tests (sVNTs) against a variant-specific in-house VSV-pseudotyped virus neutralization test (pVNT) as the gold standard. Results: Employing variant-specific assays recapitulated the immune escape functions of virus variants. Conspicuously, antibody reactivity against Alpha and Omicron BA.1 and BA.5 was strikingly poor in IBD patient sera post-initial vaccination compared to healthy individuals. A comparison of the diagnostic performance of assays with the pVNT revealed that identification of patients with inadequate humoral responses by CMIA and sVNT may require adjustments to cut-off values and end-point titration of sera. Following adaptation of cut-off values, patient sera exhibited reduced reactivity against all tested variants. The assay panel used substantiated the impact of anti-TNF therapy in IBD patients as to reduced strength, function, and breadth of the immune response to several SARS-CoV-2 variants. The immune response measured following the second vaccination was comparable to the antibody response observed in healthy individuals following the first vaccination. Conclusion: Variant-specific sVNTs and pVNTs have the potential to serve as valuable tools for evaluating the efficacy of adapted vaccines and to inform clinical interventions in the care of immunosuppressed patients. Anti-TNF-treated individuals with antibody levels below the optimized CMIA threshold should be considered for early booster vaccination and/or close immunological monitoring. Full article

Background/Objectives: Severe acute respiratory syndrome (SARS-CoV-2) has had a profound global impact and continues to represent a health challenge worldwide. The durability of SARS-CoV-2 vaccine responses in pediatric inflammatory bowel disease (PIBD) patients receiving biologic therapies is unknown. This study aimed to quantify SARS-CoV-2 antibody responses post vaccination in these immunosuppressed patients over 12 months. Methods: Prospective study comparing antibody responses against SARS-CoV-2 spike protein at 1, 3, 6, and 12 months in PIBD patients aged 5–18 years treated with anti-tumor necrosis factor alpha (anti-TNF) therapies with or without an immunomodulator (IM) versus vedolizumab. Results: Between 1 May 2021 and 1 May 2022, 194 participants on anti-TNF monotherapy (n = 78), anti-TNF with IM (n = 83), vedolizumab (n = 15), and steroids (n = 18) were recruited. Anti-SARS-CoV-2 spike levels increased after the first vaccine and were further boosted 1 month after the second dose. Linear mixed-effects modelling showed antibody waning over time (effect difference −2509 IgG AU/mL per week [95%CI: −4998–−20, p = 0.048]), counterbalanced by booster doses (effect difference 184,138 IgG AU/mL per additional vaccine dose [95%CI: 138,342–229,934, p < 0.001]). Receiving anti-TNF therapy contributed to reduced antibody responses compared to vedolizumab (anti-TNF monotherapy effect difference: −212,640 [95%CI: −336,928–−88,351] p = 0.001; anti-TNF with IM: −151,880 [95%CI: −277,309–−26,451] p = 0.018). Seroconversion and breakthrough infection rates were similar between groups, and all infections were mild, without hospitalizations. Conclusions: Although SARS-CoV-2 antibody responses were attenuated in PIBD patients receiving anti-TNF therapy compared with vedolizumab, this did not impact protection, as seroconversion and breakthrough infection rates were similar, with no hospitalizations. These data reinforce the importance of updating vaccines and, in particular, SARS-CoV-2 vaccines in immunosuppressed PIBD patients on advanced therapies. Full article

Background: The 2019 coronavirus disease (COVID-19) pandemic had a severe impact on frail, end-stage kidney disease (ESKD) patients, either on dialysis or transplanted, with a high mortality rate in the early waves. Vaccination against SARS-CoV-2 with mRNA vaccines has led to reduced hospitalization and mortality rates in the general population and ESKD patients. Neutralizing antibodies (NAbs) are a valuable correlate of protection after vaccination, and IgG anti-spike antibodies are considered a surrogate marker of protection. Methods: This study investigated the correlates of protection brought by NAb and anti-spike IgG antibodies against SARS-CoV-2 wild-type Wuhan strain and variants of concern in a cohort of 128 French patients on dialysis after vaccination with the BNT162b2 mRNA vaccine. The correlate was assessed using Receiver Operating Characteristic curves. Results: The level of protection for IgG anti-spike antibodies was set at 917 BAU/mL for the original Wuhan strain and 980 BAU/mL and 1450 BAU/mL, respectively, for the Delta and Omicron BA.1 variants. Conclusions: The level of protection can be regularly monitored by measuring IgG anti-spike antibody concentrations to allow tailored boosters of SARS-CoV-2 vaccination in this frail and immunocompromised ESKD population. Full article

mRNA vaccines have demonstrated considerable efficacy and safety against SARS-CoV-2, limiting the pandemic burden worldwide. The emergence of new variants of concern and the decline in neutralizing activity observed several weeks post-vaccination reinforced the call for repeated mRNA vaccination. We and others have shown that vaccine efficacy does not exclusively rely on antibody neutralizing activites; Fc-effector functions play an important role as well. However, it is well known that long-term exposure and repeated antigen stimulation elicit the IgG4 subclass of antibodies, which are inefficient at mediating Fc-effector functions. In this regard, recent studies highlighted concerns about IgG4 induction by mRNA vaccines. Here, we explored the impact of repeated mRNA vaccination on IgG4 induction and its impact on Fc-effector functions. We observed anti-Spike IgG4 elicitation after three doses of mRNA vaccine; the antibody levels further increased with additional doses. Vaccine-elicited IgG4 preferentially bound the ancestral D614G Spike. We also observed that Breakthrough Infection (BTI) after several doses of vaccine strongly increased IgG1 levels but had no impact on IgG4 levels, thereby improving Fc-effector functions. Finally, we observed that elderly donors vaccinated with Moderna mRNA vaccines elicited higher IgG4 levels and presented lower Fc-effector functions than donors vaccinated with the Pfizer mRNA vaccine. Altogether, our results highlight the importance of monitoring the IgG subclasses elicited by vaccination. Full article

Background/Objectives: The aim of our study was to investigate B cell and T cell responses in people with multiple sclerosis (PwMS) treated with ocrelizumab, a humanized anti-CD20 antibody, who were vaccinated with second and/or booster doses of various vaccine brands against COVID-19. Additionally, we detected the outcomes related to COVID-19 in PwMS after vaccination, based on follow-up for at least 12 months. Methods: We enrolled 91 PwMS on ocrelizumab and 42 healthy controls (HCs) in a prospective, single-center study, conducted at the Clinic of Neurology, UCCS, between January 2022 and October 2024. The serological responses were measured using the spike receptor-binding domain (RBD) Architect SARS-CoV-2 IgG Quant kit (Abbot), and cellular responses were measured by quantifying IFN-γ secretion in blood incubated with SARS-CoV-2 antigens. Results: A total of 58.2% (53/91) of PwMS on ocrelizumab and 100% of the HCs (42/42) were seropositive after a second or booster vaccination (p < 0.001), irrespective of the vaccine brand received. Anti-spike antibody levels were significantly lower in PwMS on ocrelizumab compared to the HCs (p < 0.001), again irrespective of the vaccine type. Interferon-γ responses were detected in 95.6% of the PwMS receiving ocrelizumab therapy and 97.6% of HCs after vaccination (p = 0.570). In our cohort, PCR-confirmed SARS-CoV-2 infections after vaccination occurred in a similar proportion of the PwMS (45/91, 49.5%) and HCs (15/32, 46.9%) (p = 0.139). Most of the PwMS (36/45, 79.2%) and HCs (13/15, 87.8%) had COVID-19 of mild severity. Conclusions: PwMS treated with ocrelizumab developed diminished humoral and robust cellular responses following two and three SARS-CoV-2 vaccinations. The obtained immunity after SARS-CoV-2 vaccination may translate into lower incidence and severity of COVID-19. Full article

An increase in immunoglobulin G4 (IgG4) levels is typically associated with immunological tolerance states and develops after prolonged exposure to antigens. Accordingly, IgG4 is considered an anti-inflammatory antibody with a limited ability to trigger efficient immune responses. Additionally, IgG4 reduces allergic reactions by blocking immunoglobulin E (IgE) activity. In the case of COVID-19, it has been reported that the repeated administration of some vaccines induces high IgG4 levels. The latest research data have revealed a surprising IgE anti-receptor binding domain response after both natural infection and several SARS-CoV-2 vaccines. The presence of IgG4 and IgE in COVID-19 disease suggests that the virus may induce an “allergic-like” response to evade immune surveillance, leading to a shift from T helper 1 (Th1) to T helper 2 (Th2) cells, which promotes tolerance to the virus and potentially contributes to chronic infection. The spike protein from vaccines could also induce such a response. Interestingly, “allergen-like” epitopes and IgE responses have been reported for other viruses, such as influenza, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV). The impact of this viral-induced tolerance will be discussed, concerning long COVID and the protective efficacy of vaccines. Full article

Background/Objectives: This study mapped antibody dynamics across three COVID-19 vaccination rounds (primary course, first, and second booster with BNT162b2) in Belgian nursing home residents (NHRs). Methods: Within a national SARS-CoV-2 serosurveillance study (February 2021–September 2022) across Belgian nursing homes, dried blood spots were collected, on which anti-spike SARS-CoV-2 IgG antibodies were quantified by ELISA in international units/mL (IU/mL). Sociodemographic data were collected at the study start and infection history and vaccination data at each sampling round. Results: Infection-naïve NHRs had low antibody levels after primary course vaccination (geometric mean concentration (GMC) 292 IU/mL, 95% confidence interval (95% CI): 197–432), but increased tenfold after first booster (GMC 2168 IU/mL, 95% CI: 1554–3027). While antibodies among NHRs significantly declined within six months after primary vaccination (p < 0.0001), they remained stable for nine months post-booster (p > 0.05). Among primary vaccine non-responders, 92% (95% CI: 82–97%) developed antibodies after the first booster (GMC 594 IU/mL, 95% CI: 416–849), though tenfold lower than initial responders (GMC 4642 IU/mL, 95% CI: 3577–6022). Conclusions: These findings demonstrate that NHRs require tailored vaccination, prioritizing repeated immunization to improve serological outcomes in poor responders such as infection-naive NHRs. Regular immune monitoring could aid in implementing evidence-based vaccine strategies, ensuring optimal protection for vulnerable populations against SARS-CoV-2 and other infectious threats. Full article

Background: Lipid nanoparticles (LNPs) have proven effective in delivering RNA-based modalities. Rapid approval of the COVID-19 vaccines highlights the promise of LNPs as a delivery platform for nucleic acid-based therapies and vaccines. Nevertheless, improved LNP designs are needed to advance next-generation vaccines and other gene therapies toward greater clinical success. Lipid components and LNP formulation excipients play a central role in biodistribution, immunogenicity, and stability. Therefore, it is important to understand, identify, and assess the appropriate lipid components for developing a safe and effective formulation. Herein, this study focused on developing a novel Polysorbate-80 (PS-80)-based LNP. We hypothesized that substituting conventional linear PEG-lipids with PS-80, a widely used, biocompatible injectable surfactant featuring a branched PEG-like structure, may change the LNPs biodistribution pattern and enhance long-term stability. By leveraging PS-80’s unique structural properties, this study aimed to develop an mRNA-LNP platform with improved extrahepatic delivery and robust freeze/thaw tolerance. Methods: We employed a stepwise optimization to establish both the lipid composition and formulation buffer to yield a stable, high-performing PS-80-based SARS-CoV-2 mRNA-LNP (SC2-PS80 LNP). We compared phosphate- versus tris-based buffers for long-term stability, examined multiple lipid ratios, and evaluated the impact of incorporating PS-80 (a branched PEG-lipid) on in vivo biodistribution. Various analytical assays were performed to assess particle size, encapsulation efficiency, mRNA purity, and in vitro potency of the developed formulation and a humanized mouse model was used to measure its immunogenicity over six months of storage at −80 °C. Results: Replacing the standard 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol-2000 (PEG-DMG) lipid with PS-80 increased spleen-specific expression of the mRNA-LNPs after intramuscular injection. Formulating in a tris-sucrose-salt (TSS) buffer preserved the LNP’s physicochemical properties and in vitro potency over six months at −80 °C, whereas a conventional PBS-sucrose (PSS) buffer was less protective under frozen conditions. Notably, TSS-based SC2-PS80 LNPs elicited potent humoral immunity in mice, including high anti-spike IgG titers and robust pseudovirus neutralization, comparable to freshly prepared formulations. Conclusions: A PS-80-based mRNA-LNP platform formulated in TSS buffer confers improved extrahepatic delivery, long-term frozen stability, and strong immunogenicity against SARS-CoV-2 following six months. These findings offer a promising pathway for the design of next-generation mRNA vaccines and therapeutics with enhanced stability and clinical potential. Full article

Background: As COVID-19 has become an epidemic, we conducted an open-label study aimed to identify immunogenicity and reactogenicity of boosters of the BNT162b2 vaccine in a real-world cohort of long-survivor metastatic lung cancer patients (LS-mLC pts). Methods and Analysis: According to the timing of the booster dose (BD) and SARS-CoV-2 infection (Cov-I) during anticancer treatment (ACT), between October 2021 and February 2022, we prospectively enrolled 166 cancer patients into five parallel cohorts. The primary endpoints were seroprevalence of IgG Anti-spike-RBD (anti-S IgG) at two pre-defined timepoints (T1: +30–90 days after BD; T2: +6 months +/− 4 weeks after BD). As an exploratory endpoint, we compared the median pre-vaccination value of four cytokines (IL-6, IL-2R, TNF-α, IL-10) with post-BD values in immunotherapy-treated pts (IO-pts). Results: The anti-S IgG seropositivity rate was 100% at T1 and 98.8% at T2. After 6 months, hybrid immunisation was associated with a higher median anti-S IgG titre compared to vaccine-alone-induced seroconversion (p < 0.0001). In uninfected pts, the median anti-S IgG titre was significantly lower in IO-pts compared to non-IO-pts (p = 0.02); no difference was found when comparing myelosuppressive or not ACT. Among the 68 IO-pts, 5 pts (7.3%) showed a significant increase (≥1.5 fold) of at least two cytokines in post-BD samples, without reporting ir-AEs. Conclusions: Boosters of the COVID-19 mRNA vaccine were effective and safe. In IO-pts without recent Cov-I, additional BDs should be considered to prolong serological immunity. Full article

This study investigates the impact of immunological and clinical factors on COVID-19 outcomes among unvaccinated individuals. A cohort of 42 unvaccinated patients admitted to an intensive care unit was analyzed, focusing on age, comorbidities, inflammatory cytokines (IL-6, TNF-α), and anti-SARS-CoV-2 spike protein antibody levels (IgG) to assess their influence on hospital stay duration, recovery time, complications, and mortality rates. The findings revealed that advanced age, cardiovascular disease, and elevated pro-inflammatory cytokines significantly heightened the risks of severe complications and mortality. Conversely, low IgG levels correlated with prolonged hospital stays and slower recovery. Multivariate analysis identified high IL-6 and TNF-α levels as strong predictors of adverse outcomes. This research emphasizes the need for the early monitoring of cytokines and targeted management strategies to mitigate the impact of COVID-19, especially among high-risk unvaccinated populations. Full article

Background/Objectives: Virus-like particles (VLPs) represent an attractive platform for delivering vaccine formulations, combining a high biosafety profile with a potent immune-stimulatory ability. VLPs are non-infectious, non-replicating, self-assembling nanostructures that can be exploited to efficiently expose membrane-tethered glycoproteins such as the SARS-CoV-2 Spike (S) protein, the main target of approved preventive vaccines. Here, we describe the development and preclinical validation of Simian Immunodeficiency Virus (SIV)-based GFP-labeled VLPs displaying S from the B.1.617.2 (Delta) variant (VLP/S-Delta) for inducing persistent anti-SARS-CoV-2 neutralizing antibodies (nAbs) and S-specific T cell responses in mice. Methods: SIV-derived VLP/S-Delta were produced by co-transfecting a plasmid expressing SIVGag-GFP, required for VLP assembly and quantification by flow virometry, a plasmid encoding the Delta S protein deleted in the cytoplasmic tail (CT), to improve membrane binding, and a VSV.G-expressing plasmid, to enhance VLP uptake. Recovered VLPs were titrated by flow virometry and characterized in vitro by transmission electron microscopy (TEM) and confocal microscopy (CLSM). BALB/c mice were immunized intramuscularly with VLP/S-Delta following a prime–boost regimen, and humoral and cellular immune responses were assessed. Results: VLP/S-Delta were efficiently pseudotyped with CT-truncated S-Delta. After BALB/c priming, VLP/S-Delta elicited both specific anti-RBD IgGs and anti-Delta nAbs that significantly increased after the boost and were maintained over time. The prime–boost vaccination induced similar levels of cross-nAbs against the ancestral Wuhan-Hu-1 strain as well as cross-nAbs against Omicron BA.1, BA.2 and BA.4/5 VoCs, albeit at lower levels. Moreover, immunization with VLP/S-Delta induced S-specific IFNγ-producing T cells. Conclusions: These data suggest that SIV-based VLPs are an appropriate delivery system for the elicitation of efficient and sustained humoral and cellular immunity in mice, paving the way for further improvements in the immunogen design to enhance the quality and breadth of immune responses against different viral glycoproteins. Full article

Background: Vaccines against COVID-19 target the spike protein. There is minimal information on longitudinal COVID-19 immune profiling in recovered versus naïve and vaccinated versus non-vaccinated healthcare workers (HCWs). Methods: This is a prospective longitudinal observational cohort of pediatric HCWs (pHCWs) conducted during 2020–2022 at an academic center, exploring the impact of COVID-19 vaccination on immunoglobulin G (IgG) antibody titers over time and cross-reactivity with other coronaviruses, including SARS-CoV-1, MERS-CoV, and seasonal coronaviruses (HCoV-HKU1 and HCoV-OC43). Results: A total of 642 pHCWs initially enrolled, and 337 participants had repeat IgG titers measured post-vaccine and post-booster. Most participants were female, median age range of 31–40 years. Anti-spike was higher in all vaccinated individuals versus non-vaccinated (p < 0.0001) and naïve versus infected (p < 0.0001). A single dose of vaccine was sufficient to attain maximum titers in recovered participants versus naïve who received both doses of vaccine. Anti-spike titers dropped significantly at 9 months after the primary series, whereas sustained anti-spike titers were observed at 9 months post-booster. Conclusions: All vaccinated pHCWs developed antibodies to spike. COVID-19 infection and/or vaccination yielded antibodies that cross-reacted to SARS-CoV-1, MERS-CoV, HCoV-HKU1, and HCoV-OC43. Anti-spike titers were more durable post-booster compared to the primary series. Longitudinal immune profiling of COVID-19 responses provides vital data to shape public health policies, optimize vaccine strategies, and strengthen pandemic preparedness. Full article