Neuropathological lesions in intravenous BCG-stimulated K18-hACE2 mice challenged with SARS-CoV-2.

In the wake of the COVID-19 pandemic caused by SARS-CoV-2, questions emerged about the potential effects of Bacillus Calmette-Guérin (BCG) vaccine on the immune response to SARS-CoV-2 infection, including the neurodegenerative diseases it may contribute to. To explore this, an experimental study was carried out in BCG-stimulated and non-stimulated k18-hACE2 mice challenged with SARS-CoV-2. Viral loads in tissues determined by RT-qPCR, histopathology in brain and lungs, immunohistochemical study in brain (IHC) as well as mortality rates, clinical signs and plasma inflammatory and coagulation biomarkers were assessed. Our results showed BCG-SARS-CoV-2 challenged mice presented higher viral loads in the brain and an increased frequency of neuroinvasion, with the greatest differences observed between groups at 3-4 days post-infection (dpi). Histopathological examination showed a higher severity of brain lesions in BCG-SARS-CoV-2 challenged mice, mainly consisting of neuroinflammation, increased glial cell population and neuronal degeneration, from 5 dpi onwards. This group also presented higher interstitial pneumonia and vascular thrombosis in lungs (3-4 dpi), BCG-SARS-CoV-2 mice showed higher values for TNF-α and D-dimer values, while iNOS values were higher in SARS-CoV-2 mice at 3-4 dpi. Results presented in this study indicate that BCG stimulation could have intensified the inflammatory and neurodegenerative lesions promoting virus neuroinvasion and dissemination in this experimental model. Although k18-hACE2 mice show higher hACE2 expression and neurodissemination, this study suggests that, although the benefits of BCG on enhancing heterologous protection against pathogens and tumour cells have been broadly demonstrated, potential adverse outcomes due to the non-specific effects of BCG should be considered.

Prevalence and Severity of COVID-19 among Pediatric Patients with Atopy: A Cross-sectional Study in Kerman, Southeast Iran.

The tragic COVID-19 pandemic affected many children worldwide. Among the factors that may influence the course of viral infections including COVID-19, it is still uncertain whether atopy has a protective or predisposing role. The study aims to address the knowledge gap by investigating the prevalence and severity of COVID-19 among atopic children in Kerman, in 2022. A descriptive-analytical cross-sectional study on children with a history of atopy was performed in Kerman Medical University. Demographic information, type of atopy (including allergic rhinitis, Hyper-Reactive Airway Disease (HRAD) or asthma, eczema, urticaria, anaphylaxis, and food allergy), history of COVID-19 infection, and disease severity were recorded. A total of 1007 children and adolescents, (boys: 56.4%, girls: 43.6%, age:5.61±2.64 years) were included in the study. History of COVID-19 infection was positive in 53.5%, with 75.9% of the cases exhibiting mild disease severity. The frequency of atopies was HRAD or asthma (67.2%), allergic rhinitis (42.6%), and food allergy (27.4%). The frequency of COVID-19 cases was significantly higher among patients with HRAD or asthma, whereas it was significantly lower among those with food allergies, anaphylaxis, and eczema. Among atopic individuals, COVID-19 severity was significantly lower in those with allergic rhinitis, while the opposite trend was observed among food-allergic individuals. This study sheds light on the relationship between atopy and COVID-19 among pediatric patients. It seems specific types of atopies may influence the risk and severity of COVID-19 infection differently. A better understanding of these associations can inform clinical management and preventive measures for vulnerable pediatric populations.

Antibody Response Before and After the Booster Dose of Inactivated Corona Vaccine in Antibody Deficient Patients.

Patients with inborn errors of immunity (IEI) are among the high-risk groups regarding COVID-19. Receiving booster doses (third and fourth) in addition to the standard doses is recommended in these patients. This study investigated the antibody response before and after a booster dose of Sinopharm vaccine in IEI patients.  Thirty patients (>12 years) with antibody deficiencies, referred to Imam Khomeini Hospital and Children's Medical Center in Tehran, were enrolled in this prospective cross-sectional study. All patients were fully vaccinated with the BBIBP-CorV vaccine (2 doses of Sinopharm). Initial measurements of anti-receptor-binding domain (anti-RBD) and anti-nucleocapsid (anti-N) IgG antibody responses were conducted by enzyme-linked immunosorbent assay (ELISA). Subsequently, all patients received a booster dose of the vaccine. Four to six weeks after booster injection, the levels of antibodies were re-evaluated.  Twenty patients with common variable immunodeficiency (CVID), 7 cases with agammaglobulinemia and 3 patients with hyper IgM syndrome were studied. Anti-RBD IgG and anti-N IgG antibodies increased in all patients after the booster. Our results indicated the need of receiving booster doses of the COVID-19 vaccine in patients with antibody deficiencies, even for enhancing humoral immune response specially in patients with CVID.

TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.

Effectiveness of upper arm and breathing exercises to improve inflammatory markers in severe COVID-19 patients.

Studies have suggested associations between inflammatory markers with the severity of coronavirus disease 2019 (COVID-19). Therefore, exercises that could reduce the level of inflammatory markers might be beneficial. The aim of this study was to determine the effect of upper arm and breathing exercises on inflammatory markers such as ferritin, lactate dehydrogenase (LDH), and C-reactive protein (CRP) in severe COVID-19 patients. A quasi-experimental with pre-test and post-test control group design was conducted among severe COVID-19 aged 18-70 years old, with or without comorbidities. Baseline data of inflammatory markers (ferritin, LDH, and CRP) were measured before the exercises and repeated post-exercise. The upper arm and breathing exercises were performed for ten days, twice a day (morning and evening) for ten minutes. A paired Student t-test was used to assess the changes in the inflammatory markers' levels. Our data indicated that levels of ferritin and CRP were not significantly different between pre- and post-exercise. However, the level of LDH decreased significantly from 481.35 U/L to 331.80 U/L (p=0.001). This study highlights that pulmonary rehabilitation exercises might be beneficial to enhance the recovery process in severe COVID-19 patients.

Relationship between neutrophil-lymphocyte ratio and platelet-lymphocyte ratio with the severity of COVID-19.

Coronavirus disease 2019 (COVID-19) is an infectious disease that spreads rapidly causing a high case fatality rate in vulnerable populations. Neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) are known to be inflammatory biomarkers in certain infections. The aim of this study was to determine the relationship between NLR and PLR with the severity of COVID-19. A cross-sectional study was conducted at Tabanan Regency General Hospital, Bali, Indonesia, from January 2021 to December 2022. All patients included in the study tested positive for COVID-19 by real-time polymerase chain reaction (RT-PCR), aged 18-50 years with no comorbid. Laboratory examinations were carried out on admission. The patients were categorized into two groups based on the severity: moderate and severe/critical. The Mann-Whitney test was used to determine the association between NLR and PLR with the severity of COVID-19. A total of 104 patients were included in the study, the majority of COVID-19 patients had moderate (77.9%) severity. The average NLR was 5.8 and the PLR was 21.7. There was a significant relationship between NLR (p=0.002) and PLR (p=0.001) with the severity of COVID-19. The defined cut-off values of NLR and PLR were ≥3.8 and ≥106, yielding sensitivities of 95% and 70%, and specificities of 74% and 50%, respectively. This study highlights the promising role of NLR and PLR as predictive biomarkers to assess COVID-19 severity.

Gene expression signatures in blood from a West African sepsis cohort define host response phenotypes.

Our limited understanding of the pathophysiological mechanisms that operate during sepsis is an obstacle to rational treatment and clinical trial design. There is a critical lack of data from low- and middle-income countries where the sepsis burden is increased which inhibits generalized strategies for therapeutic intervention. Here we perform RNA sequencing of whole blood to investigate longitudinal host response to sepsis in a Ghanaian cohort. Data dimensional reduction reveals dynamic gene expression patterns that describe cell type-specific molecular phenotypes including a dysregulated myeloid compartment shared between sepsis and COVID-19. The gene expression signatures reported here define a landscape of host response to sepsis that supports interventions via targeting immunophenotypes to improve outcomes.

Neutralizing antibody responses and cellular responses against SARS-CoV-2 Omicron subvariants after mRNA SARS-CoV-2 vaccination in kidney transplant recipients.

Although the mRNA SARS-CoV-2 vaccine has improved the mortality rate in the general population, its efficacy against rapidly mutating virus strains, especially in kidney transplant recipients, remains unclear. We examined the anti-SARS-CoV-2 spike protein IgG antibody and neutralizing antibody titers and cellular immunity against B.1.1, BA.1, and BA.5 antigens in 73 uninfected kidney recipients and 16 uninfected healthy controls who received three doses of an mRNA SARS-CoV-2 vaccine. The IgG antibody titers were significantly lower in recipients than in healthy controls. Similarly, neutralizing antibody titers against three viral variants were significantly lower in recipients. When the virus was mutated, the neutralizing antibody titers decreased significantly in both groups. In cellular immunity analysis, the number of spike-specific CD8 + non-naïve T cells against three variants significantly decreased in recipients. Conversely, the frequency of spike-specific Th2 CD4 + T-cells in recipients was higher than that in healthy controls. Nineteen recipients and six healthy controls also received a bivalent omicron-containing booster vaccine, leading to increase IgG and neutralizing antibody titers in both groups. After that, eleven recipients and five healthy controls received XBB.1.5 monovalent vaccines, increasing the neutralizing antibody titers against not only XBB.1.5, but also EG.5.1 and BA.2.86 antigens in kidney recipients. Although kidney recipients did not gain sufficient immunity against Omicron BA.5 with the third dose of vaccine, humoral response against mutant SARS-CoV-2 lineages significantly increased after bivalent Omicron-containing booster vaccine and the XBB.1.5 monovalent vaccine. Therefore, it is important for kidney recipients to continue to administer updated vaccines.

Efficient signal sequence of mRNA vaccines enhances the antigen expression to expand the immune protection against viral infection.

The signal sequence played a crucial role in the efficacy of mRNA vaccines against virus pandemic by influencing antigen translation. However, limited research had been conducted to compare and analyze the specific mechanisms involved. In this study, a novel approach was introduced by substituting the signal sequence of the mRNA antigen to enhance its immune response. Computational simulations demonstrated that various signal peptides differed in their binding capacities with the signal recognition particle (SRP) 54 M subunit, which positively correlated with antigen translation efficiency. Our data revealed that the signal sequences of tPA and IL-6-modified receptor binding domain (RBD) mRNA vaccines sequentially led to higher antigen expression and elicited more robust humoral and cellular immune protection against the SARS-CoV-2 compared to the original signal sequence. By highlighting the importance of the signal sequence, this research provided a foundational and safe approach for ongoing modifications in signal sequence-antigen design, aiming to optimize the efficacy of mRNA vaccines.

Functional and structural investigation of a broadly neutralizing SARS-CoV-2 antibody.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.

SARS-CoV-2 omicron BA.2.87.1 exhibits higher susceptibility to serum neutralization than EG.5.1 and JN.1.

As SARS-CoV-2 continues to spread and mutate, tracking the viral evolutionary trajectory and understanding the functional consequences of its mutations remain crucial. Here, we characterized the antibody evasion, ACE2 receptor engagement, and viral infectivity of the highly mutated SARS-CoV-2 Omicron subvariant BA.2.87.1. Compared with other Omicron subvariants, including EG.5.1 and the current predominant JN.1, BA.2.87.1 exhibits less immune evasion, reduced viral receptor engagement, and comparable infectivity in Calu-3 lung cells. Intriguingly, two large deletions (Δ15-26 and Δ136-146) in the N-terminal domain (NTD) of the spike protein facilitate subtly increased antibody evasion but significantly diminish viral infectivity. Collectively, our data support the announcement by the USA CDC that the public health risk posed by BA.2.87.1 appears to be low.

Infection- and Vaccine-Induced SARS-CoV-2 Seroprevalence, Japan, 2023.

We assessed SARS-CoV-2 seroprevalence in Japan during July-August 2023, with a focus on 2 key age groups, 0-15 and >80 years. We estimated overall seroprevalence of 45.3% for nucleocapsid antibodies and 95.4% for spike antibodies and found notable maternally derived spike antibodies in infants 6-11 months of age (90.0%).

Mosaic RBD nanoparticle elicits immunodominant antibody responses across sarbecoviruses.

Nanoparticle vaccines displaying mosaic receptor-binding domains (RBDs) or spike (S) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or other sarbecoviruses are used in preparedness against potential zoonotic outbreaks. Here, we describe a self-assembling nanoparticle using lumazine synthase (LuS) as the scaffold to display RBDs from different sarbecoviruses. Mosaic nanoparticles induce sarbecovirus cross-neutralizing antibodies comparable to a nanoparticle cocktail. We find mosaic nanoparticles elicit a B cell receptor repertoire using an immunodominant germline gene pair of IGHV14-3:IGKV14-111. Most of the tested IGHV14-3:IGKV14-111 monoclonal antibodies (mAbs) are broadly cross-reactive to clade 1a, 1b, and 3 sarbecoviruses. Using mAb competition and cryo-electron microscopy, we determine that a representative IGHV14-3:IGKV14-111 mAb, M2-7, binds to a conserved epitope on the RBD, largely overlapping with the pan-sarbecovirus mAb S2H97. This suggests mosaic nanoparticles expand B cell recognition of the common epitopes shared by different clades of sarbecoviruses. These results provide immunological insights into the cross-reactive responses elicited by mosaic nanoparticles against sarbecoviruses.

The Potential Mechanisms of Arrhythmia in Coronavirus disease-2019.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) leads to coronavirus disease-2019 (COVID-19) which can cause severe cardiovascular complications including myocardial injury, arrhythmias, acute coronary syndrome and others. Among these complications, arrhythmias are considered serious and life-threatening. Although arrhythmias have been associated with factors such as direct virus invasion leading to myocardial injury, myocarditis, immune response disorder, cytokine storms, myocardial ischemia/hypoxia, electrolyte abnormalities, intravascular volume imbalances, drug interactions, side effects of COVID-19 vaccines and autonomic nervous system dysfunction, the exact mechanisms of arrhythmic complications in patients with COVID-19 are complex and not well understood. In the present review, the literature was extensively searched to investigate the potential mechanisms of arrhythmias in patients with COVID-19. The aim of the current review is to provide clinicians with a comprehensive foundation for the prevention and treatment of arrhythmias associated with long COVID-19.

SARS-CoV-2 active infection and antibodies amongst health personnel during the outbreak in Cameroon: Strengthening the health system for response to future public health emergencies.

BACKGROUND: Health personnel (HP) are on the frontlines during response to public health emergencies like COVID-19. This risk of exposure suggests the need for safety in responding to any pandemic. Therefore, to ascertain the rate of SARS-CoV-2 infection and immunity, and their determinants amongst HP become relevant. METHODS: A cross sectional health facility-based study was carried-out amongst HP in the Centre Region of Cameroon from 1st February to 30th June 2021. Characteristics and access to preventive tools were collected using face-to-face administered questionnaire. Nasopharyngeal swabs and whole blood were collected for PCR, IgG and IgM testing respectively. STATA version 17 software was used for data analysis. Determinants of COVID-19 infection were explored by estimating crude and adjusted Odd Ratio. RESULTS: Out of 510 HP reached, 458 were enrolled with mean age of 35 (±10) years. Thirty-four (7.4%) were PCR-positive to SARS-CoV-2 with 73.5% being clinicians versus 9 (26.4%) non-clinicians (p = 0.05). Sero-positivity to SARS-CoV-2 IgG/IgM was 40.2% (184/458), with 84.2% being clinicians versus 29 (15.8%) non-clinicians (p = 0.733). Amongst the 34 HP with PCR-positivity, 16 (47%) had no antibodies, while, 15 (44%) were IgG only. An estimate of HP (43.7%) had at least an evidence of PCR, IgG or IgM contact to COVID-19. Determinants of PCR-positivity was being clinical staff (AOR = 0.29, P = 0.039); and that of IgG/IgM were being non clinical staff (AOR = 0.41, p = 0.018) and regular use of face masks (AOR = 0.44, p = 0.001). HP trained on IPC (24%) were mainly from peripheral level (74.7%, p = 0.002). CONCLUSION: Active infections were within the range of pandemic control (<10%). However, around two-fifths of participants have had contact with the virus, indicating that HP remains a population at risk of COVID-19 and other similarly-transmitted epidemic prone diseases, and also an important source of transmission. There is need of vaccine to achieve protectiveness, and optimal response also requires capacity building to improve the health system when challenged by a future pandemic.

A novel immunofluorescent test system for SARS-CoV-2 detection in infected cells.

Highly variable pandemic coronavirus SARS-CoV-2, which causes the hazardous COVID-19 infection, has been persistent in the human population since late 2019. A prompt assessment of individual and herd immunity against the infection can be accomplished by using rapid tests to determine antiviral antibody levels. The microneutralization assay (MN) is one of the most widely used diagnostic methods that has been proposed to assess the qualitative and quantitative characteristics of virus-specific humoral immunity in COVID-19 convalescents or vaccine recipients. However, some aspects of the assay, such as sensitivity and time cost, need improvement. Here, we developed an express test, which may be potentially used in clinical practice for the assessment of serum-caused SARS-CoV-2 inhibition in infected cell cultures. It implies the detection and counting of coronaviral fluorescent-forming units (FFU) and includes two sequentially used developing components: biotinylated mouse monoclonal antibodies against the recombinant N protein of SARS-CoV-2 (B.1) and the recombinant EGFP-streptavidin fusion protein. Due to the universal specificity of the antibodies, our analytical tool is suitable for the detection of various strains of SARS-CoV-2 when determining both the infectious titer of viruses and the titer of serum virus-neutralizing antibodies. The developed two-component test system is characterized by high sensitivity, a reduced number of analytic stages and low assay cost, as well as by flexibility, since it may be modified for detection of other pathogens using the appropriate antibodies.

Dynamic heterogeneity in COVID-19: Insights from a mathematical model.

Critical illness, such as severe COVID-19, is heterogenous in presentation and treatment response. However, it remains possible that clinical course may be influenced by dynamic and/or random events such that similar patients subject to similar injuries may yet follow different trajectories. We deployed a mechanistic mathematical model of COVID-19 to determine the range of possible clinical courses after SARS-CoV-2 infection, which may follow from specific changes in viral properties, immune properties, treatment modality and random external factors such as initial viral load. We find that treatment efficacy and baseline patient or viral features are not the sole determinant of outcome. We found patients with enhanced innate or adaptive immune responses can experience poor viral control, resolution of infection or non-infectious inflammatory injury depending on treatment efficacy and initial viral load. Hypoxemia may result from poor viral control or ongoing inflammation despite effective viral control. Adaptive immune responses may be inhibited by very early effective therapy, resulting in viral load rebound after cessation of therapy. Our model suggests individual disease course may be influenced by the interaction between external and patient-intrinsic factors. These data have implications for the reproducibility of clinical trial cohorts and timing of optimal treatment.

Distinct T cell responsiveness to different COVID-19 vaccines and cross-reactivity to SARS-CoV-2 variants with age and CMV status.

Introduction: Accumulating evidence indicates the importance of T cell immunity in vaccination-induced protection against severe COVID-19 disease, especially against SARS-CoV-2 Variants-of-Concern (VOCs) that more readily escape from recognition by neutralizing antibodies. However, there is limited knowledge on the T cell responses across different age groups and the impact of CMV status after primary and booster vaccination with different vaccine combinations. Moreover, it remains unclear whether age has an effect on the ability of T cells to cross-react against VOCs. Methods: Therefore, we interrogated the Spike-specific T cell responses in healthy adults of the Dutch population across different ages, whom received different vaccine types for the primary series and/or booster vaccination, using IFNÉ£ ELISpot. Cells were stimulated with overlapping peptide pools of the ancestral Spike protein and different VOCs. Results: Robust Spike-specific T cell responses were detected in the vast majority of participants upon the primary vaccination series, regardless of the vaccine type (i.e. BNT162b2, mRNA-1273, ChAdOx1 nCoV-19, or Ad26.COV2.S). Clearly, in the 70+ age group, responses were overall lower and showed more variation compared to younger age groups. Only in CMV-seropositive older adults (>70y) there was a significant inverse relation of age with T cell responses. Although T cell responses increased in all age groups after booster vaccination, Spike-specific T cell frequencies remained lower in the 70+ age group. Regardless of age or CMV status, primary mRNA-1273 vaccination followed by BNT162b2 booster vaccination showed limited booster effect compared to the BNT162b2/BNT162b2 or BNT162b2/mRNA-1273 primary-booster regimen. A modest reduction in cross-reactivity to the Alpha, Delta and Omicron BA.1, but not the Beta or Gamma variant, was observed after primary vaccination. Discussion: Together, this study shows that age, CMV status, but also the primary-booster vaccination regimen influence the height of the vaccination-induced Spike-specific T cell response, but did not impact the VOC cross-reactivity.

Characterizing the antigenic evolution of pandemic influenza A (H1N1) pdm09 from 2009 to 2023.

The H1N1pdm09 virus has been a persistent threat to public health since the 2009 pandemic. Particularly, since the relaxation of COVID-19 pandemic mitigation measures, the influenza virus and SARS-CoV-2 have been concurrently prevalent worldwide. To determine the antigenic evolution pattern of H1N1pdm09 and develop preventive countermeasures, we collected influenza sequence data and immunological data to establish a new antigenic evolution analysis framework. A machine learning model (XGBoost, accuracy = 0.86, area under the receiver operating characteristic curve = 0.89) was constructed using epitopes, physicochemical properties, receptor binding sites, and glycosylation sites as features to predict the antigenic similarity relationships between influenza strains. An antigenic correlation network was constructed, and the Markov clustering algorithm was used to identify antigenic clusters. Subsequently, the antigenic evolution pattern of H1N1pdm09 was analyzed at the global and regional scales across three continents. We found that H1N1pdm09 evolved into around five antigenic clusters between 2009 and 2023 and that their antigenic evolution trajectories were characterized by cocirculation of multiple clusters, low-level persistence of former dominant clusters, and local heterogeneity of cluster circulations. Furthermore, compared with the seasonal H1N1 virus, the potential cluster-transition determining sites of H1N1pdm09 were restricted to epitopes Sa and Sb. This study demonstrated the effectiveness of machine learning methods for characterizing antigenic evolution of viruses, developed a specific model to rapidly identify H1N1pdm09 antigenic variants, and elucidated their evolutionary patterns. Our findings may provide valuable support for the implementation of effective surveillance strategies and targeted prevention efforts to mitigate the impact of H1N1pdm09.