Search Results (655)

Background: Emerging immune-evasive viral variants threaten the efficacy of current vaccines, underscoring the need for strategies that elicit broad and durable protection. Heterologous prime–boost regimens combining distinct vaccine platforms can enhance humoral and cellular immunity through complementary mechanisms. Methods: Using an intramuscular immunization scheme aligned with clinical vaccination practice, CD-1 mice received homologous or heterologous prime–boost regimens combining a replication-deficient Orf virus (Parapoxvirus orf, ORFV)-based spike vaccine (ORFV-S) with the licensed adjuvanted recombinant protein vaccine VidPrevtyn Beta. Spike-specific humoral and cellular immune responses were assessed. Results: ORFV-S alone induced potent and broad spike-specific IgG responses and achieved the strongest ACE2-binding inhibition across variants of concern. ORFV-S priming followed by VidPrevtyn Beta boosting markedly enhanced the magnitude and cross-variant breadth of antibody responses compared with homologous protein vaccination. Both homologous ORFV-S and heterologous regimens incorporating ORFV-S elicited strong CD4⁺ and CD8⁺ T-cell responses, whereas VidPrevtyn Beta alone induced only modest T-cell activity, demonstrating that ORFV-S effectively complements protein-based vaccines. Conclusions: The ORFV-S vector represents a potent vaccine platform capable of inducing broad humoral and cellular immunity. Its use in heterologous prime–boost combinations enhances both antibody magnitude and breadth beyond homologous protein vaccination, supporting its application in vaccination strategies against evolving viral pathogens. Full article

Background/Objective: Despite available SARS-CoV-2 vaccines, coverage gaps persist due to unequal distribution and limited access. Microarray patches offer a promising solution to address these challenges, providing a safer and easier-to-use alternative. We present a randomised, double-blind Phase I clinical trial evaluating the SARS-CoV-2 spike protein subunit vaccine, HexaPro, delivered via a high-density microarray patch (HD-MAP). Methods: Forty-four healthy adults aged 18–50 years were assigned to receive either 0 µg, 15 µg, or 45 µg of HexaPro via the HD-MAP, with the primary objective of assessing safety and tolerability. Results: The HD-MAP HexaPro vaccine was found to be safe and well tolerated, with only mild adverse events reported. Following vaccination significant increases in spike-specific IgG titers were observed by 7 days and remained stable through day 90. This IgG response effectively neutralised multiple SARS-CoV-2 variants. Additionally, the HexaPro HD-MAP was stable for up to 12 months at 40 °C. Conclusions: These findings support the continued clinical development of HD-MAPs as an alternative vaccination strategy. Full article

Background: Memory B cells (B_mem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of B_mem is at the core of most vaccination strategies, the assessment of antigen-specific B_mem is highly informative for forecasting and profiling the elicited B cell immune response. Methods: The two prevalent techniques used to detect antigen-specific B_mem cells at single-cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these assay systems has not yet been reported using the same starting PBMC material in a blinded fashion to test all three assays simultaneously. Results: These three assay systems were run in parallel to detect SARS-CoV-2 Wuhan-1 strain Spike-specific IgG⁺ B_mem in peripheral blood mononuclear cell (PBMC) samples obtained from well-defined cohorts comprising pre-COVID-19 era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMCs isolated in 2024 (the experimental group). Each assay was able to discern Spike-exposed individuals from naïve , with ImmunoSpot suggesting superior sensitivity and specificity. ImmunoSpot and flow cytometry results were closely correlated. Conclusions: The study demonstrates that all three assays are suited for the detection of specific B_mem in antigen-primed individuals when such B_mem occur in the mid- to high-frequency range, and that they broadly concur. Strengths and weaknesses of the three test systems are discussed. Full article

Background: Antigen format strongly influences the immunogenicity of gene-based vaccines. Full-length Spike is widely used in licensed COVID-19 vaccines, while truncated subunits such as S1 or the receptor-binding domain (RBD) may simplify vector design but risk reduced potency. We aimed to compare these antigen formats in an AAV9 delivery platform. Methods: BALB/c mice were immunized intramuscularly with recombinant AAV9 encoding full-length Spike, S1, or RBD at doses of 1 × 10¹⁰ or 1 × 10¹¹ viral genomes. Immune responses were assessed by serology, virus neutralization, T-cell profiling, and histopathology. Results: All constructs expressed antigen in vitro and in vivo. Only full-length Spike elicited robust neutralizing antibodies at both doses, with titers rising significantly by week 12. High-dose RBD induced neutralization in a minority of animals, whereas S1 failed to do so. Antigen-specific IgG responses scaled with insert length (Spike > S1 > RBD). Cellular immunity was dominated by CD8⁺ effector memory T cells, strongest in the Spike group, which also induced measurable CD4⁺ responses. Local transient myositis was observed at the injection site but resolved by week 24, with no systemic pathology. Conclusions: Full-length Spike outperforms truncated subunits in the AAV context, highlighting antigen structure as a critical factor for next-generation coronavirus vaccine design. Full article

Background: Healthcare workers (HCWs) are at high risk of breakthrough SARS-CoV-2 infections despite complete vaccination schedules. There are gaps in our understanding of the specific antibody isotypes and cytokine profiles produced during an infection following vaccination. In this study, we evaluated SARS-CoV-2⁻specific antibody isotypes and their association with cytokine production in HCWs with breakthrough infections. Methods: Serum samples from 114 HCWs were analyzed for antibody isotypes against the nucleoprotein (NCP) and the receptor binding domain (RBD) of the spike protein, as well as for a panel of 13 cytokines. Results: Vaccinated SARS-CoV-2⁺ HCWs showed a higher prevalence of anti-SARS-CoV-2 antibodies against NCP (IgM = 93.8%, IgG = 93.8%, IgA = 28.1%) and RBD (IgM = 46.9%, IgG = 100%, IgA = 90.6%). A specific IgM response to NCP was more frequent in vaccinated SARS-CoV-2⁺ individuals, whereas IgA responses were predominantly specific for RBD. Both pro- and anti-inflammatory cytokines were elevated in vaccinated HCWs with breakthrough infections compared with unvaccinated and uninfected individuals. Interestingly, infected IgG+ HCWs with IgM specific for both NCP and RBD exhibited significantly higher IL-8, IL-6, TNF-α, IFN-γ, IL-2, IL-10, and TGF-β concentrations. Conclusion. Our data show that breakthrough infections in vaccinated HCWs induce a robust pro-and anti-inflammatory cytokine profile, which is associated with a broader IgM response directed against both NCP and RBD. Full article

Background: During the SARS-CoV-2 pandemic, many women were infected or received vaccinations against the virus before or during their pregnancy. Little is known about the possible consequences of vaccination or infection on obstetric outcomes, as well as antibody levels against other infectious agents, such as the TORCH pathogens. Methods: A total of 136 pregnant women were included in our study between March 2022 and February 2024. The concentrations of antibodies against nucleocapsid (NCP), the spike protein of SARS-CoV-2, as well as IgG and IgM antibodies against TORCH, were assessed in the maternal and umbilical cord blood. Results: The patients were grouped into the following categories according to responses given in the questionnaire and antibody titer: controls (neither infected nor vaccinated; N = 17), infected only (N = 35), vaccinated only (N = 21), acutely infected (N = 15), and both vaccinated and experienced a COVID-19 infection (N = 47). No differences between the groups in terms of pregnancy outcomes were found. The presence of IgG antibodies against NCP or spike protein in maternal blood was dependent on the patient’s vaccination status or previous infection, correlating with that in cord blood. The level of maternal IgG against spike protein correlated negatively with TORCH antibodies. Conclusions: The present study demonstrates the infection- and vaccination-dependent formation of SARS-CoV-2-specific antibodies in the mother and their transfer to the unborn child. Further studies are necessary to investigate the interaction between SARS-CoV-2-specific antibodies and antibodies formed by infection (e.g., CMV) or vaccination against other pathogens in the mother and transmitted transplacentally to the unborn child. Full article

Introduction: The COVID-19 pandemic posed additional challenges for this vulnerable population, such as Sjögren’s disease (SjD), underscoring the need for effective and safe vaccination strategies. Objective: To evaluate the immunogenicity and safety of COVID-19 vaccines in patients with SjD. Methods: This prospective, observational, longitudinal study included SjD patients from the SAFER cohort. Immunogenicity was assessed via anti-spike IgG (IgG-S) titers using chemiluminescence reported as geometric mean titers (GMT) and fold increase in GMT (FI-GMT). Disease activity was evaluated using the ESSDAI score. Adverse events and COVID-19 infections were also monitored. Assessments were conducted at four time points: pre-first dose (T1), pre-second dose (T2), pre-booster (T3), and four weeks post-booster (T4). Primary vaccination involved ChAdOx1 nCoV-19 or inactivated vaccine (CoronaVac), and boosters were either homologous (ChAdOx1 nCoV-19) or heterologous (BNT162b2). Results: Among 51 participants (mean age 46 years; 90% female), 41% had comorbidities and 27% (n = 14/51) were highly immunosuppressed. Among those 73% (n = 37/51) under low immunosuppression, n = 8/51 (13%) were not using any medication. At baseline, 11% (n = 4/35) showed moderate/high disease activity, which decreased to 6.5% (n = 2/31) at T4. Primary vaccination was ChAdOx1 in 94% (n = 48/51) and CoronaVac in 6% (n = 3/51); 73% (n = 37/51) received heterologous and 27% (n = 14/51) homologous boosters. COVID-19 infection post-booster occurred in 20% (n = 10/51). Seroconversion rates reached nearly 100% across all medication subgroups except for biologic users, who showed delayed but stable seroconversion by T4. IgG-S titers increased progressively through T4. Primary immunization induced an ascending GMT in both vaccine types. At T4, the GMT was significantly higher in the BNT162b2 group (2148.03 [1452.05–3155.84]; p < 0.001; 95% CI) than in the ChAdOx1 group (324.29 [107.92–974.48]; p < 0.001; 95% CI); the fold-increase in immune response was six times greater with BNT162b2 (5.98 [2.97–12.03]; p = 0.001; 95% CI). Seroconversion was 100% in the heterologous group versus 83% in the homologous group (p > 0.01). Those with prior infection showed significantly higher titers, particularly at T2 and T3 (p < 0.001 for T1–T3). Adverse events were mild and not statistically significant. Multivariate regression confirmed BNT162b2 as an independent factor for higher antibody titers. Conclusion: COVID-19 vaccination in patients with SjD was safe and induced high anti-spike antibody titers and seropositivity. Heterologous boosting, particularly with BNT162b2, demonstrated superior immunogenicity. No association was found between vaccination and SjD disease flares or worsening activity. Full article

Background/Objectives: The anti-spike (S) SARS-CoV-2 antibodies confer neutralizing properties and their concentration may be related to COVID-19 protection. Anti-nucleocapsid (N) SARS-CoV-2 antibodies in mRNA COVID-19 vaccine recipients indicate infection. The aim of this study was to analyze the anti-S and anti-N titers 3 years after COVID-19 vaccination. Methods: Ninety-nine vaccinated healthcare workers provided blood samples in 2024 and filled out questionnaires about their COVID-19 history and boosters acceptance. Anti-spike and anti-nucleocapsid IgG were assessed with commercially available immunoassays, DiaSorin’s SARS-CoV-2 TrimericS IgG and Abbott’s SARS-CoV-2 IgG, respectively. Results: Three years after the primary COVID-19 vaccination, the anti-S SARS-CoV-2 antibody concentration was still high. However, it dropped in comparison to the data obtained a year before (3600 vs. 2040 BAU/mL), possibly due to the lack of boosters. In contrast, the percentage of anti-N seropositive individuals grew from 34% two years after vaccination to 40.4% after three years. Subjects with SARS-CoV-2 infection within a year prior to the antibody measurements had statistically significantly higher median anti-S concentrations than those with tentatively no contact with SARS-CoV-2 (2940 vs. 1930 BAU/mL). Conclusions: Overall, our data indicates that although the booster vaccinations’ acceptance decreases, the circulating SARS-CoV-2 stimulates humoral immunity, resulting in high anti-S antibody concentrations even three years after the vaccination. Full article

Background/Objectives: Although the U.S. Food and Drug Administration (FDA) has approved one antiviral treatment and authorized others for emergency use, there is no fully effective antiviral therapy for coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Assays detecting virus-specific immunoglobulins (Ig) or nucleic acids in large-scale epidemiological, vaccine, and drug development studies remain limited due to high costs, reagent accessibility, and cumbersome protocols. Methods: A multiplex bead-based assay was developed to simultaneously detect human IgM, IgG, and IgA antibodies against the SARS-CoV-2 spike receptor binding domain (RBD) in serum using flow cytometry. Assay performance was evaluated for sensitivity, specificity, reproducibility, and cross-reactivity and compared to another immunoassay platform. Results: The assay enabled simultaneous measurement of three antibody isotypes across 624 samples within 2 h. Intra-plate coefficients of variation (CVs) ranged from 3.16 to 6.71%, and inter-plate CVs ranged from 3.33 to 5.49%, demonstrating high reproducibility. The platform also quantified background noise from nonspecific binding, facilitating straightforward data interpretation. Conclusions: This novel, flexible multiplex bead-based assay utilizing a well-established platform provides a rapid and reproducible approach for detecting SARS-CoV-2-specific antibodies. Its high throughput capacity and low variability make it well suited for large-scale epidemiological, vaccine, and therapeutic studies. The platform’s adaptability further supports application to other infectious diseases, offering an ideal tool for broad immunological surveillance. Full article

Complex matrix of vegetable severely interferes with enzyme-linked immunosorbent assay (ELISA) accuracy, limiting its application in parathion residue detection. This study investigated the interference mechanism of vegetable matrix, including chlorophyll, perilla protein, glucose, fructose, and sucrose, on ELISA. Furthermore, we validated the vegetable matrix interference on parathion residue ELISA by comparing the matrix interference index (I_m) and recovery rate of vegetable samples before and after acetic acid-treatment. The results demonstrate that the addition of vegetable matrix significantly interferes with ELISA, with the antibody–IgG-HRP binding being subject to the most pronounced interference. Compared to the I_m (16–26%) of non-acetic acid treatment, the I_m (10–13%) was significantly reduced after the acetic acid treatment. Concomitantly, spiked recovery experiments of acid-treated samples yielded satisfactory average recovery rate (80–113%) as the matrix interference was minimized. The findings of this study provide valuable insights into the mechanism of vegetable matrix interference on ELISA. Full article

Background/Objectives: Understanding the durability of immunity induced by mRNA COVID-19 vaccines, especially in individuals with chronic health conditions, remains essential for guiding booster strategies. We conducted a longitudinal study to evaluate humoral and cellular immune responses up to 21 months after a primary two-dose BNT162b2 vaccination followed by a booster, either homologous (BNT162b2) or heterologous (mRNA-1273). Methods: Twenty-eight adults, mostly with chronic conditions, were assessed at approximately 9, 12 and 21 months post-primary vaccination. Serum anti-trimeric Spike IgG levels were quantified, and peripheral blood mononuclear cells were analyzed at 21 months for Spike-specific memory B-cell and T-cell responses by flow cytometry. Results: Participants were stratified by booster type, prior SARS-CoV-2 infection and health status. Anti-Spike IgG persisted in all participants but declined over time. The heterologous mRNA-1273 booster induced higher antibody titers at 9 months, while the homologous BNT162b2 booster led to more sustained antibody levels and higher frequencies of Spike-specific memory B cells at 21 months. Prior infection significantly enhanced antibody titers, particularly in homologous booster recipients. Surprisingly, individuals with chronic health conditions exhibited equal or higher antibody levels compared to healthy participants at all time points. At 21 months, robust Spike-specific class-switched memory B cells and polyfunctional CD4⁺ and CD8⁺ T-cell responses were detected. Conclusions: These findings demonstrate that BNT162b2 vaccination elicits durable, multi-layered immunity lasting nearly two years, even in individuals with chronic conditions, and support the use of both homologous and heterologous mRNA boosters to sustain protection in diverse populations. Full article

Introduction (Objectives): This study aimed to evaluate the serological response to a fourth dose of mRNA COVID-19 vaccine in patients with conditions that confer a high risk of severe disease, particularly those with high-level immunosuppression. Methods: An observational study was conducted at the Ramón y Cajal University Hospital between February and August 2022. Adults (≥18 years) with high-risk conditions who had received four doses of either BNT162b2 or mRNA-1273 were included. Anti-spike IgG levels were measured ≥14 days post-vaccination. An adequate response was defined as an antibody concentration ≥260 BAU/mL. Results: A total of 943 patients were analyzed; 846 (89.7%) achieved an adequate response. In the bivariate analysis, patients aged 60–74 years had a higher risk of inadequate response compared to those aged 18–39 years (OR 1.824 vs. OR 0.257). Female sex was associated with a higher risk of inadequate response (OR 1.522; 95% CI: 0.974–2.371). In multivariable logistic regression, patients with high immunosuppression had a higher, though not statistically significant, risk of inadequate response compared with those without. Discussion: Our findings are consistent with international evidence suggesting that age and certain clinical factors reduce vaccine immunogenicity. The observed paradoxical effect of sex could reflect the higher prevalence of aggressive immunosuppressive therapies among women in the study cohort. Conclusions: Most immunosuppressed patients achieved seroconversion after the fourth dose. These results underscore the need for tailored vaccination strategies and additional measures in highly immunosuppressed subgroups. Full article

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to evolve, with mutations leading to the emergence of new variants. JN.1, a subvariant of omicron BA.2.86, has demonstrated marked immune escape and is now included in updated vaccine formulations. While reduced sensitivity has been reported for antibody assays using ancestral spike protein subunits to detect omicron-induced responses, the performance of full-length spike-based assays against omicron sublineages remains unclear. We aimed to compare the sensitivity of ELISA and Luminex assays using full-length spike proteins from the ancestral Wuhan strain and the JN.1 variant. Methods: Wuhan and JN.1 full-length spike protein constructs were designed and expressed in Expi293F mammalian cells. In-house ELISAs based on previously validated protocols were used to measure anti-spike IgG levels. Additionally, a Luminex-based assay for anti-spike antibody detection was developed and validated. Both assays were applied to the following sample groups: pre-pandemic samples (designated “gold standard negatives”); PCR confirmed 2020 positives (“gold standard wildtype positives”); PCR confirmed 2024 positives (“gold standard omicron positives”); 2022 vaccinated individuals with verbal confirmed infection (“gold standard hybrid positives”); and 2024 household samples (“unknowns”). Results: Wuhan spike protein showed a sensitivity of 100% (95% CI: 0.88–1.0) in detecting omicron-specific antibodies using gold standard omicron positives with JN.1 spike protein as a reference assay. Overall, across all samples, in ELISA, the Wuhan antigen had a sensitivity of 0.93 (95% CI: 0.89–0.95) and a specificity of 0.98 (95% CI: 0.94–0.99). The JN.1 antigen showed a sensitivity of 0.91 (95% CI: 0.87–0.94) and a specificity of 0.97 (95% CI: 0.93–0.99). In Luminex, sensitivity was 0.95 (95% CI: 0.91–0.97) for Wuhan and 0.94 (95% CI: 0.91–0.96) for JN.1. Specificity for both antigens in Luminex was 0.98 (95% CI: 0.94–0.99). Conclusions: Both ELISA and Luminex assays showed comparable sensitivity and specificity for both Wuhan and JN.1 antigens, indicating that mutations in the JN.1 variant do not significantly impact assay performance. This suggests preserved antigenic recognition across variants. Full article

Background and Objectives: SARS-CoV-2 infection initiates at mucosal surfaces, and mucosal immunity may influence the nature and severity of infection. Little is known about the induction of mucosal immunity by vaccination in COVID-19 convalescents. Methods: Sera from 205 healthcare workers were collected one month after the first Covishield vaccination and 1/3/6 months after the second vaccination, while paired sera and stimulated whole-mouth fluid (SWMF) was collected 1/3/6 months after the third vaccination (N = 10) and at 0/30/90 days after a COVID-19 episode (N = 8). Anti-SARS-CoV-2 spike antibody detection by ECLIA/ELISA and cytokine detection by ELISA/CBA were performed. Results: One month post-second vaccination, serum antibodies had increased significantly (6-fold) in the COVID-19-naïve group (CNG) but declined (1.5-fold) in the previously COVID-19-exposed group (CEG), who already had high antibody titres. The serum regulatory cytokine IL-10 levels were higher after three antigen exposures (p = 0.0002). New infections (breakthrough infections—BTIs) or reinfections (RIs) with asymptomatic/mild disease occurred in 44% of the CNG and 27% of the CEG (p < 0.01). The mucosal cytokine IL-17 levels were significantly higher in the CEG. Salivary IgG/IgA and secretory IgA antibodies were detectable both after vaccination and COVID-19. Innate cytokines (MIG, MCP-1, IL-8, IL-1β) were higher and sustained in SWMF in contrast to serum. Conclusions: Two vaccinations in the CNG resulted in an antibody boost, but the second vaccination in the CEG induced antibody anergy. Serum/mucosal antibodies declined by six months after vaccination, but the rapid increase at subsequent exposures were indicative of a good T cell/B cell memory response to SARS-CoV-2. A higher percentage of BTI among the CNG than RI among the CEG may indicate better protection due to higher antibody responses in the latter group. Full article

Background: Vaccination against SARS-CoV-2 has been pivotal in controlling the COVID-19 pandemic. However, understanding vaccine-induced immunity in immunocompromised individuals remains critical, particularly how prior exposure to other coronaviruses modulates immune responses. The influence of previous infections with endemic human coronaviruses (HCoVs), such as OC43, on SARS-CoV-2 immunity is not fully understood. This study evaluates antibody responses to COVID-19 vaccination in hemodialysis patients (HD), transplant recipients (TR), and healthy controls (CO), accounting for prior SARS-CoV-2 infection and baseline human coronavirus (HCoV) reactivity. Methods: We obtained longitudinal antibody measurements from 70 subjects (CO: n = 33; HD: n = 13; TR: n = 24) and assessed antibody kinetics across multiple post-vaccination time points using multivariate linear mixed modeling (MLMM). Results: Limited but measurable cross-reactivity was observed between SARS-CoV-2 and endemic HCoVs, particularly the $\beta$-coronavirus OC43. Pre-existing immunity in healthy individuals modestly enhanced vaccine-induced anti-spike (S) IgG responses, supported by post-vaccination increases in SARS-CoV-2 IgG. Prior SARS-CoV-2 infection significantly influenced anti-S and nucleocapsid (N) IgG responses but had limited impact on endemic HCoVs responses. Vaccine type and immune status significantly affected antibody kinetics. mRNA vaccination (BNT162b2) elicited stronger and more durable SARS-CoV-2 anti-S IgG responses than the inactivated CoronaVac vaccine, especially in immunocompetent individuals. Immunocompromised groups showed delayed or attenuated responses, with modest anti-S IgG cross-reactive boosting. Elevated anti-N IgG in CoronaVac recipients raised questions about its origin—infection or vaccine effects. MLMM identified key immunological and clinical predictors of antibody responses, emphasizing the critical role of host immune history. Conclusions: These findings highlight a constrained but meaningful role for HCoV cross-reactivity in SARS-CoV-2 immunity and vaccine responsiveness, underscore the need for infection markers unaffected by vaccination, and support development of broadly protective pan-coronavirus vaccines and tailored strategies for at-risk populations. Full article