Search Results (576)

Background: Individuals with autoimmune disease, inflammatory bowel disease (IBD), and transplants face a higher risk of severe COVID-19 and breakthrough hospitalizations. It is essential to understand the magnitude and durability of vaccine-induced humoral immune response in these populations. Methods: We evaluated SARS-CoV-2 spike IgG antibody levels and avidity in autoimmune, IBD, transplant, and healthy cohorts following multiple COVID-19 mRNA vaccine doses. Serum samples were collected approximately 1 month (8–52 days) and 6 months (158–202 days) post-vaccination. Antibody levels and avidity were measured using validated ELISA and chaotropic-based avidity assays. Results: Individuals with IBD and individuals with autoimmune disease, especially systemic autoimmune disease, exhibited lower antibody levels and avidity compared with healthy individuals at certain doses and time points. Transplant recipients demonstrated substantial impairments in both antibody levels and avidity, with avidity reduced across all doses and time points. Significantly lower avidity levels were observed in transplant recipients, suggesting challenges in developing or maintaining antibody quality. For example, geometric mean anti-spike IgG levels were substantially lower in transplant recipients than in healthy individuals at both 1 month (635 vs. 7685 BAU/mL; p < 0.0001) and 6 months (1146 vs. 3277 BAU/mL; p = 0.0057) post third dose. Similarly, transplant recipients had lower antibody avidity than healthy individuals after the third dose, with geometric mean AI80 values of 4.5 M vs. 5.5 M at 1 month (p < 0.0001) and 4.6 M vs. 5.4 M at 6 months (p < 0.0001). Age, sex, and vaccine manufacturer may further influence humoral immune responses in the transplant cohort. Conclusions: Vaccine-induced humoral immunity varies across autoimmune, IBD, and transplant cohorts, with the most persistent impairment observed in transplant recipients across vaccine dose groups and at both post-vaccination time points. These findings reveal immune response patterns that may guide future studies evaluating vaccination schedules, immune monitoring, and clinical outcomes in these populations. Full article

Background/Objectives: Waning immunity after primary COVID-19 vaccination supports evaluation of additional doses. HXP-GPOVac is an egg-based, inactivated Newcastle disease virus (NDV)-vectored vaccine expressing a prefusion-stabilized SARS-CoV-2 HexaPro spike antigen. We evaluated the safety, tolerability, and immunogenicity of a single additional 10 µg dose of HXP-GPOVac administered to adults previously primed with two doses of either HXP-GPOVac or BNT162b2. Methods: Study GPO NDV-HXP-S 203 was an open-label phase II extension enrolling adults (18–75 years) who previously completed a two-dose primary series in Study 202 with either HXP-GPOVac or BNT162b2 (Pfizer–BioNTech; Comirnaty). All participants received a single additional 10 µg intramuscular dose of HXP-GPOVac ≥ 6 months after their second primary dose. Solicited local/systemic adverse events (AEs) were recorded for 7 days, unsolicited AEs through Day 28, and serious AEs (SAEs) and adverse events of special interest (AESIs) throughout follow-up. Neutralizing antibody titers (pseudovirus 50% neutralization titer, NT₅₀) and anti-spike IgG (BAU/mL) were assessed pre-dose (Day 1) and post-vaccination through 12 months; a predefined subset underwent IFN-γ and IL-5 ELISpot. SARS-CoV-2 infection during follow-up was assessed using anti-nucleocapsid (anti-N) IgG. Symptomatic COVID-19 was identified through symptom-reported, symptom-triggered RT-PCR testing; sequencing was performed when feasible. Results: All 219 participants received HXP-GPOVac (167 primed with HXP-GPOVac and 52 with BNT162b2). Any solicited local reaction occurred in 22.2% (37/167) of HXP-GPOVac-primed and 26.9% (14/52) of BNT162b2-primed participants; any solicited systemic reaction occurred in 10.8% (18/167) and 13.5% (7/52), respectively. No vaccine-related unsolicited AEs or AESIs were reported. Three deaths occurred during the 12-month follow-up; one (a sudden cardiac death in an HXP-GPOVac-primed participant) was assessed by the safety medical team as possibly related to vaccination, and two were assessed as not related. Neutralizing antibody GMTs increased from 46.33 at baseline to 1569.04 at Day 15 in HXP-GPOVac-primed participants and from 77.25 to 841.34 in BNT162b2-primed participants; corresponding SCRs were 78.8% and 76.9%. Anti-spike IgG GMCs increased from 48.79 to 1480.14 BAU/mL and from 194.48 to 1547.88 BAU/mL, respectively. Responses declined over time but remained above baseline through 12 months. In the cellular immunity subset, post-vaccination IFN-γ responses increased, with comparatively modest IL-5 responses and no pattern suggestive of Th2 predominance. Conclusions: A single additional dose of HXP-GPOVac administered ≥6 months after primary vaccination with HXP-GPOVac or BNT162b2 was generally well tolerated and elicited robust recall humoral responses, with supportive findings of cellular immunity. Trial registration: Thai Clinical Trials Registry, TCTR20230213001. Full article

Messenger RNA (mRNA) vaccines represent a transformative platform in vaccinology, with applications extending beyond SARS-CoV-2 to other infectious diseases and cancer immunotherapy. However, patients with solid tumors receiving active anticancer treatment were largely underrepresented in pivotal vaccination trials, limiting understanding of vaccine-induced immunity in this population. In this prospective exploratory study, we assessed humoral and cellular immune responses after two doses of SARS-CoV-2 mRNA vaccines in 39 patients with solid tumors undergoing active treatment. Blood samples were collected before vaccination and approximately two months after the second vaccine dose, prior to the next treatment cycle. Anti-spike IgG, neutralizing antibodies, receptor-binding domain (RBD) levels, interleukin-6 (IL-6), hematological parameters, immune cell subsets, T-cell differentiation, and early apoptosis in αβ and γδ T-cell subsets were analyzed. Vaccination induced a robust humoral response, with high post-vaccination anti-spike IgG levels (median 988.69 BAU/mL), 97.44% seropositivity, 96.88% true seroconversion among baseline IgG−/NAb− patients, and strong neutralizing antibody activity (median 85.73%). Hematological parameters and IL-6 levels remained broadly stable, suggesting no detectable increase in systemic inflammation during the study period. Cellular analyses identified a reduction in peripheral CD19+ B-cell frequencies and decreased early apoptosis, particularly in CD8+ T cells and CD3+CD56+ NKT-like cells. Although changes in T-cell frequencies and differentiation profiles were also observed, these findings were attenuated after exclusion of participants with possible prior SARS-CoV-2 exposure and should be interpreted as exploratory. Overall, these results show that patients with solid tumors receiving active treatment can mount robust humoral responses to SARS-CoV-2 mRNA vaccination and suggest measurable post-vaccination changes in lymphocyte dynamics, including reduced early T-cell apoptosis. Full article

Background: Despite the circulation of evolutionarily related cold-causing coronaviruses (CCCs) in the pre-COVID era, most individuals lacked pre-existing serum IgG and/or class-switched memory B cell (B_mem) reactivity for the SARS-CoV-2 Spike (S) glycoprotein expressed by the ancestral Wuhan-Hu-1 (WH1) strain. Subsequent priming of the immune system through natural infection or prophylactic COVID-19 mRNA vaccination successfully generated robust B_mem responses against the WH1-S antigen, along with eliciting cross-reactivity for the future Omicron (BA.1) variant responsible for breakthrough infections (BTIs). However, to what extent immunological imprinting of B_mem towards the WH1-S antigen detrimentally constrains the elicitation of variant-specific antibody responses following subsequent booster vaccinations or BTIs—a phenomena referred to as “original antigenic sin”—remains an unresolved and open question. Methods: Using ImmunoSpot^®, we evaluated peripheral blood mononuclear cells (PBMCs) from defined human cohorts for IgG⁺ ASC reactivity against Spike proteins representing CCCs and SARS-CoV-2. Additionally, we developed a novel dual-label inverted FluoroSpot assay to distinguish between strain-specific and cross-reactive IgG⁺ ASCs recognizing epitopes in the receptor binding domain (RBD) of SARS-CoV-2 Omicron variants. Results: Our data demonstrate a lack of appreciable back-boosting of IgG⁺ B_mem recognizing structurally conserved epitopes shared between CCCs and SARS-CoV-2. Moreover, we found evidence for immunological imprinting and the preferential expansion of B_mem recognizing cross-reactive epitopes in the RBD following BTI. Nevertheless, Omicron strain-specific B_mem were detected in PBMC donors collected in 2025. Conclusions: Our novel inverted dual-label FluoroSpot methodology evidenced preferential expansion of cross-reactive B_mem following breakthrough SARS-CoV-2 infection and supports the influence of original antigenic sin shaping the recall response. Moreover, the inverted dual-label assay provides a highly flexible and easily implementable technique for distinguishing between strain-specific and cross-reactive B cell responses and has broad applications in translational vaccine research against pathogens that undergo antigenic drift. Full article

Background: The COVID-19 pandemic exposed critical gaps in pandemic preparedness and highlighted the need for vaccine platforms capable of rapid adaptation. Outer membrane vesicle (OMV)-based platforms utilizing vesicles derived from genetically detoxified Neisseria meningitidis serogroup B (Nm-nOMV) represent a promising plug-and-play approach. Methods: This Phase I, first-in-human, randomized, double-blind, placebo- and OMV-controlled trial, evaluated safety, tolerability, and immunogenicity of intranasally administered OMVs combined with SARS-CoV-2 Spike protein in healthy SARS-CoV-2 seropositive adults aged 18–55 years. Forty participants were enrolled across two cohorts: a low-dose cohort receiving 140 μg OMV/70 μg Spike (OMV + Spike, n = 13; OMV alone, n= 3; Placebo, n = 5) and a high-dose cohort receiving 280 μg of OMV/140 μg of Spike (OMV + Spike, n = 13; OMV alone, n = 3; Placebo, n = 3), administered on Days 1 and 22. Safety was assessed through adverse events, vital signs, laboratory parameters, ECG, and pulse oximetry. Immunogenicity was evaluated via systemic SARS-CoV-2 neutralizing antibodies, antigen-specific antibodies (IgG and IgA), and mucosal antibodies (IgA in nasal wash). Results: Intranasal administration of OMVs combined with SARS-CoV-2 Spike protein was safe, well-tolerated, and immunogenic. No serious adverse events were reported, and adverse events were predominantly mild and transient. Dose-dependent increases in systemic and mucosal immune responses were observed, with statistically significant enhanced serum IgG and nasal wash IgA antibodies in the high-dose group. Conclusions: The current clinical data confirm key aspects of the preclinical profile, which demonstrate the potential of the Nm-nOMV platform as a strong adjuvant for mucosal vaccines. These findings support the broader application of the Nm-nOMV vaccine platform in pandemic preparedness. Full article

Introduction: From 12 March 2020, when the first cases of COVID-19 were registered in Kosovo, to 9 March 2023, there were a total of 273,310 reported cases of COVID-19 and 3211 reported deaths in Kosovo (CFR: 1.17%). Health care workers (HCWs) have been at a higher risk of contracting SARS-CoV-2 infection; nevertheless, data on seroprevalence of SARS-CoV-2 antibodies among HCWs in Kosovo are very limited. Methodology: A cross-sectional serology study with 1654 healthcare professionals throughout Kosovo was conducted to determine the presence of antibodies against the spike antigen of SARS-CoV-2. In addition, a structured questionnaire was administered to study participants to obtain basic demographic data, and information on prior infection and COVID-19 vaccination status. Results: Antibodies against the spike antigen of SARS-CoV-2 were detected in almost all (99.8%) HCWs that participated in the study. The average antibody titer was 8030.8 AU/mL in women and 9533.7 AU/mL in men. Sixty-four percent of HCWs in this study reported prior infection with SARS-CoV-2, 6% of whom were hospitalized. Over 98% of study participants had received SARS-CoV-2 vaccination. Conclusions: Almost all HCWs participating in the study had antibodies against the spike antigen of SARS-CoV-2. This is most probably the result of the high COVID-19 vaccination rate in Kosovo as well as infection with SARS-CoV-2. Full article

Background: The dynamics of humoral immune responses following primary and booster COVID-19 vaccinations are crucial to understand in order to optimize vaccination strategies. This study evaluates the magnitude and durability of SARS-CoV-2-specific IgG antibody responses across different vaccines in a large cohort of Bangladeshi adults. Methods: A total of 6300 adults from nine hospitals across eight divisions of Bangladesh were enrolled. Participants received two primary doses of either ChAdOx1 nCoV-19 (Covishield, Serum Institute of India, n = 2855), mRNA-1273 (Moderna, n = 578), BNT162b2 (Pfizer-BioNTech, n = 121), or Vero-cell-inactivated (Sinopharm, n = 2746) vaccines. Booster doses were administered at one-year intervals post-primary vaccination. SARS-CoV-2 spike receptor-binding domain (RBD)-specific IgG antibody responses were measured by ELISA using serum from vaccinees at multiple time points after two primary and two booster doses. Results: A total of 3745 individuals received booster 1 (third dose), with 59% receiving heterologous boosters (a different vaccine regimen than the primary doses). Only 5.5% (n = 347) of participants received a second booster one year after the first booster (among them, 99% received BNT162b2). Our results suggest that heterologous boosters with the mRNA vaccine induced higher IgG levels than homologous boosters for individuals who received primary vaccination with adenovirus vector-based ChAdOx1 nCoV-19 or a Vero-cell-inactivated vaccine. However, in those who initially received the mRNA-based vaccine, both homologous and heterologous boosters produced comparable IgG responses. Among all vaccine types, booster immunization with the Vero-cell-inactivated vaccine induced the lowest antibody responses. Longitudinal analysis demonstrated significantly high IgG levels over the 12 months following the first booster (p < 0.0001); however, IgG levels declined significantly after the second booster dose (fourth dose). Conclusions: Heterologous boosting strategies, particularly those involving mRNA vaccines, elicit stronger and more sustained IgG responses compared to a homologous booster. However, antibody waning after the second booster highlights the need for continued monitoring and potential additional vaccine strategies. Full article

Background/Objectives: In the first months of their life, infants rely on maternal antibodies for immune protection. Breastmilk is a major source of these defenses, supplying secretory IgA, IgG, and IgM that help guard mucosal surfaces against pathogens such as SARS-CoV-2. Most studies on breastmilk immunity in the context of COVID-19 have emphasized circulating monomeric IgA, rather than the multimeric secretory IgA (sIgA) that is active at mucosal barriers. This study assessed in-depth the contribution of breastmilk antibody subtypes to SARS-CoV-2 neutralization capacity and how these profiles differ following maternal COVID-19 infection versus vaccination during pregnancy or postpartum. Methods: In this prospective cohort study, breastmilk samples were collected longitudinally from individuals who had COVID-19 during pregnancy or received COVID-19 mRNA vaccination during pregnancy or postpartum. Serological assays measured IgG, IgM, systemic IgA, and secretory IgA against SARS-CoV-2 spike and nucleocapsid antigens. Results: COVID-19 infection during pregnancy resulted in significantly higher systemic and secretory IgA levels compared to vaccination. Secretory IgA demonstrated a strong correlation with neutralization capacity. Principal component analysis revealed distinct antibody profiles in COVID-19-exposed individuals versus vaccinated cohorts, with significant overlap between pregnancy and postpartum vaccination groups. Conclusions: Although both COVID-19 vaccination and disease elicit sustained COVID-19-related antibodies in breastmilk, COVID-19 infection elicits a broader and more diverse antibody response in breastmilk, specifically with a greater secretory IgA generation. These findings support the value of maternal vaccination to safely confer mucosal immunity to neonates and the need for optimized vaccine formulations for mucosal immunity. Full article

Background/Objectives: The induction of anti-SARS-CoV-2 antibodies by COVID-19 vaccination reduces morbidity and mortality, but immune responses may be compromised in people living with HIV (PLWH). The aims of the current study were to determine whether viral suppression (VS) or immune reconstitution (IR) in PLWH directly affected their ability to produce effective levels of anti-SARS-CoV-2 antibodies in mucosal secretions or blood induced by vaccination. Methods: Anti-SARS-CoV-2 spike IgG, IgA and secretory IgA (SIgA) antibodies and their avidities were measured by ELISA in HIV-negative healthy controls (HC; n = 49) and PLWH (n = 94) using stimulated oral fluid (SOF) and serum. Frequencies of CD4/CD8 T cells and their expression of exhaustion/senescence were determined by flow cytometry. Cytokine levels were measured by cytokine bead arrays. Results: We showed that higher HIV burden negatively impacted the levels of systemic and mucosal anti-SARS-CoV-2 spike IgG antibodies produced. This differential IgG antibody production was unaffected by IR status, antiretroviral therapy duration or T cell exhaustion/senescence. PLWH elicited higher anti-SARS-CoV-2 spike IgA antibodies both in peripheral blood and oral mucosa and highr secretory IgA (SIgA) antibodies in the oral mucosa. PLWH with higher HIV RNA copies elicited lower IgG avidity but the IgA avidity indices remained unaffected. PLWH expressed higher levels of innate immunity cytokines in the oral mucosa, irrespective of the HIV RNA copies. Conclusions: Significantly fewer breakthrough infections in PLWH compared with HC, along with high IgA/SIgA antibodies and increased innate immunity cytokines in the SOF, suggest a potential role for mucosal immunity in the immunopathogenesis of COVID-19. Full article

Background/Objectives: This report is an assessment of the characteristics associated with cardiovascular dysautonomia (CVD) in the context of long Coronavirus disease (COVID), which is currently inadequately characterized. Material and Methods: A retrospective cross-sectional study was performed involving 106 patients with long COVID, including 34 individuals diagnosed with CVD, among whom eight met the criteria for Postural Tachycardia Syndrome (PoTS). The variables assessed encompassed individual characteristics (e.g., age, sex, comorbidities), immunization parameters (e.g., vaccination/viral status, timing, frequency), cellular and humoral anti-Spike and anti-Nucleocapsid (Nuc) immune responses, inflammatory and allergic biomarkers, as well as an extensive panel of common autoantibodies comprising anti-nuclear antibodies, anti-central nervous system antibodies (cerebellum, brain), and anti-peripheral nervous system antibodies (gangliosides). Results: An age < 45 years, body mass index, hyperventilation syndrome as well as a higher cumulative number of antigenic contacts (vaccinations plus infections ≥ 3) and an elevated basophil count (≥0.06 G/L) were independently associated with CVD. There was no association between CVD and inflammatory markers or common autoantibodies. Patients with PoTS criteria had a strong anti-Spike cellular immune response and increased IgG anti-Nuc humoral immunity when compared with CVD and non-CVD long COVID counterparts. Conclusions: Compared to other long COVID patients, patients with long COVID-associated CVD have distinctive clinical and immunovirological features. Our results suggest the potential role of the immune response against Spike and of allergic pathways rather than humoral autoimmunity against common autoantibodies in long COVID CVD. Full article

Background/Objectives: HXP-GPOVac is a locally produced, inactivated Newcastle disease virus-based (NDV-HXP-S) COVID-19 vaccine manufactured in Thailand. This phase II trial compared its safety and immunogenicity with the mRNA vaccine BNT162b2 in adults aged 18–75 years. Methods: In this randomized, double-blind, active-controlled trial registered with the Thai Clinical Trials Registry (TCTR20220819003), 300 participants were assigned 3:1 to receive HXP-GPOVac or BNT162b2 on Days 1 and 29. Solicited adverse events (AEs) were recorded for 7 days after each dose, AEs were summarized through 28 days after each dose, and serious adverse events (SAEs), medically attended AEs (MAAEs), and adverse events of special interest (AESIs) were collected through Day 197. Humoral immunogenicity was assessed by pseudovirus 50% neutralization titers (NT₅₀) and anti-spike IgG concentrations at baseline, Day 29, Day 43, and Day 197. Seroconversion was defined as a ≥4-fold increase from baseline. A predefined subset underwent interferon-γ (IFN-γ) and interleukin-5 (IL-5) ELISpot assays to assess cell-mediated immune responses. The primary immunogenicity analysis assessed non-inferiority of HXP-GPOVac compared with BNT162b2 based on the NT₅₀ geometric mean titer ratio, with a prespecified non-inferiority margin of 0.5. Results: Solicited AEs were predominantly mild and occurred more frequently after the first dose in both groups; one or more solicited local or systemic AEs were reported by 23.7% (95% CI: 18.3–29.8) of HXP-GPOVac recipients and 44.7% (95% CI: 33.3–56.6) of BNT162b2 recipients after the first dose. AEs through 28 days after vaccination and SAEs were uncommon; MAAEs occurred in 17.0% of HXP-GPOVac recipients and 22.4% of BNT162b2 recipients, and none were considered related to vaccination. In the HXP-GPOVac group, NT₅₀ geometric mean titers increased from 5.6 at baseline to 65.5 at Day 29 and 505 at Day 43, declining to 63.6 at Day 197. Anti-spike IgG geometric mean concentrations rose from 7.5 BAU/mL at baseline to 102.7 BAU/mL at Day 29 and 514.6 BAU/mL at Day 43, decreasing to 61.0 BAU/mL at Day 197. BNT162b2 induced higher antibody levels at all time points. The NT₅₀ GMT ratio (HXP-GPOVac/BNT162b2) at Day 43 was 0.51 (95% CI: 0.39–0.67); the lower bound did not exceed the prespecified non-inferiority margin of 0.5, and non-inferiority was not established. Seroconversion rates at Day 43 were 97.6% for HXP-GPOVac and 97.1% for BNT162b2 (neutralizing antibody) and 98.6% and 97.1%, respectively (anti-spike IgG). ELISpot analyses demonstrated increased IFN-γ responses after the second dose without evidence of Th2-dominant skewing. Conclusions: HXP-GPOVac was well tolerated and induced substantial humoral and cellular immune responses, with high seroconversion rates and balanced T-cell polarization. Although absolute antibody levels were lower than those induced by BNT162b2 and the prespecified non-inferiority criterion was not met, these findings support continued evaluation of the inactivated NDV-HXP-S vaccine platform. Full article

Background: Lower respiratory tract infections remain a major cause of morbidity and mortality in infants worldwide. Newborns possess an immature immune system but acquire passive immunity through maternal antibodies transferred via the placenta (IgG) and breast milk (IgA). Maternal vaccination may enhance this protection. This study aimed to quantify antibody levels against respiratory viruses in serum and breast milk from lactating women. Methods: Serum and breast milk samples were collected from 26 lactating mothers. Antibody levels were measured using an indirect enzyme-linked immunosorbent assay (ELISA) targeting seven viral antigens: influenza A (A/Thailand, A/California), influenza B (B/Phuket, B/Austria), SARS-CoV-2 (Spike and receptor-binding domain, RBD) and RSV F pre-fusion protein. Antibody isotypes IgG, IgA and IgM were analysed. Results: Virus-specific IgG and IgA antibodies were detected in all samples. Breast milk showed the highest levels of IgA, whereas serum contained higher IgG levels. A moderate positive correlation was observed between serum and milk IgG. No correlation was found between serum IgG and milk IgA, but both levels were elevated. Conclusions: Breast milk and serum contain relatively high levels of antibodies against the tested respiratory viruses. The elevated levels of serum IgG and milk IgA indicate a coordinated defence between systemic and mucosal immunity in response to infections. The levels and correlation of specific isotypes point to the source of the antibodies: milk IgG probably originates from the blood, whereas milk IgA is produced locally. Full article

Understanding the molecular mechanisms underlying host immune responses to SARS-CoV-2 vaccination remains essential, particularly in populations with a high prevalence of obesity. In this cross-sectional study, we evaluated whether body mass index (BMI) is associated with vaccine-induced humoral immunity in a cohort from northeastern Mexico and discuss the findings within a structural immunology framework of spike antigenicity and antibody–epitope interactions. A total of 138 adults were recruited in Reynosa and Matamoros (June 2021–June 2022) and categorized as healthy weight, overweight, or obese according to BMI criteria. Serum anti-SARS-CoV-2 IgG was assessed using an ELISA-based assay, and differences across BMI groups were tested using the Kruskal–Wallis approach. Among all participants, 33.3% were classified as obese and 99.3% (137/138) were seropositive for anti-SARS-CoV-2 IgG. No significant differences in IgG levels were detected between BMI categories (p = 0.20). These results indicate that, in this Mexican cohort—sampled during a period of heterogeneous and often incomplete vaccination schedules—obesity was not associated with reduced detectable anti-SARS-CoV-2 IgG responses. Our findings support the need to integrate population-level serology with mechanistic studies that interrogate antibody quality (e.g., neutralization potency and epitope specificity) to better connect clinical determinants such as obesity with molecular correlates of protection. Full article

Background: Porcine epidemic diarrhea virus (PEDV) remains a major threat to the global swine industry, highlighting the urgent need for safe and effective next-generation vaccines. mRNA vaccines have emerged as a promising platform due to their rapid development and favorable safety profile. Objectives: This study aimed to design and perform the preliminary evaluation of a PEDV multi-epitope mRNA vaccine using an immunoinformatics-guided strategy combined with experimental validation. Methods: Immunoinformatics tools were used to identify B-cell and cytotoxic T lymphocyte (CTL) epitopes from the PEDV spike (S), membrane (M), and nucleocapsid (N) proteins. Selected epitopes were assembled into a multi-epitope antigen (E). mRNA constructs encoding S1, S2, and antigen E were synthesized via in vitro transcription and encapsulated into lipid nanoparticles (LNPs). Expression was evaluated in HEK293T cells, and immunogenicity was assessed in mice measuring antigen-specific antibody responses and cytokine levels following immunization. Results: The mRNA constructs exhibited high structural integrity and efficient intracellular translation. The LNP formulations showed good physicochemical stability and delivery efficiency. Immunization with the antigen E mRNA-LNP formulation induced significantly higher PEDV-specific IgG levels compared with control groups. Elevated cytokine levels further indicated activation of both humoral and cellular immune responses. Conclusions: This study presents a feasible workflow for the development of a PEDV multi-epitope mRNA vaccine. The antigen E construct demonstrated favorable immunogenicity in a mouse model, supporting its potential as a promising construct for further investigation and optimization. Although further studies are required to validate antigen expression at the protein level and to further characterize immune mechanisms, these findings provide preliminary evidence supporting the feasibility of multi-epitope mRNA vaccines for PEDV prevention. Full article

Background/Objective: This study is a cross-sectional investigation of long-term immune responses measured at different time intervals after COVID-19 infections, vaccinations, or combined exposure. The focus is on immune reactivity against recombinant spike (S) and nucleocapsid (N) protein antigens. Materials and Methods: Serum antibody levels were assessed up to four to four and a half years after infection or immunization, including virus-specific immunoglobulin G (IgG), IgA and IgM antibodies, as well as neutralizing antibodies against the S-protein. Cellular immunity was assessed by analyzing peripheral blood mononuclear cells (PBMC; n = 43 in first cohort, n = 32 in second cohort), including T-helper memory and cytotoxic subsets, and cytokine production after in vitro stimulation with recombinant SARS-CoV-2 proteins. A multiplex cytokine assay was used to analyze effector and regulatory immune responses. Results: Virus-specific IgG antibodies persisted for years after exposure to SARS-CoV-2, with IgG against the receptor-binding domain (RBD) correlating most strongly with neutralizing activity. Vaccinated individuals demonstrated higher IgA responses, whereas antibodies to the N-protein were associated with previous infection. No IgM antibodies were detected in any subjects, suggesting an immune response based on memory rather than ongoing infection. PBMCs from individuals with a history of both COVID-19 exposure and vaccination exhibited enhanced responsiveness, characterized by increased frequencies of memory T cells compared to vaccination alone. Stimulating with the S-protein induces higher cytokine production, including IFN-gamma, TNF-alfa, and IL-12(p70), compared with stimulation by the N-protein. Cytokines such as IL-10 and TGF-beta are also elevated, suggesting immune regulation rather than persistent inflammation. Conclusions: SARS-CoV-2 infection and vaccination are associated with persistent humoral and cellular immune responses detectable several years after exposure. Individuals with hybrid immunity exhibit broader and functionally enhanced immune reactivity, indicating more robust long-term immune memory. Future studies should focus on the long-term consequences of hybrid immunity and optimize other vaccine strategies, including recombinant antigen vaccines. Full article